IL297250A

IL297250A - Modified mini-nucleosome core proteins and use in nucleic acid delivery

Info

Publication number: IL297250A
Application number: IL297250A
Authority: IL
Original assignee: Summation Bio Inc
Priority date: 2020-04-13
Filing date: 2021-04-12
Publication date: 2022-12-01
Also published as: BR112022020771A2; EP4136235A1; CA3180120A1; CN115552013A; KR20230010036A; US20230203507A1; JP2023521999A; AU2021254697A1; MX2022012775A; WO2021211467A1

Description

MODIFIED MINI-NUCLEOSOME CORE PROTEINS AND USE IN NUCLEIC ACID DELIVERY CROSS-REFERENCE TO RELATED APPLICATIONS id="p-1" id="p-1" id="p-1" id="p-1" id="p-1"

[0001] This application claims the benefit of U.S. Provisional Application No. 63/009,124, filed on April 13, 2020, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND id="p-2" id="p-2" id="p-2" id="p-2" id="p-2"

[0002] AAV vector ares considered the current gold standard of gene therapy and have shown promise in diverse clinical trials, including clinical trials for, e.g., retinal gene therapy and systemic gene therapy in live r,CNS, and/or other tissues. With the regulatory approval of at least three different gene therapies, the field is poised for many more, so patien tscan access these life- changing treatments. However ,despite being the industry’s gold standard, AAV vector haves certain limitations. Improved and/or alternative nucleic acid delivery technologi arees needed.

SUMMARY id="p-3" id="p-3" id="p-3" id="p-3" id="p-3"

[0003] The present disclosure provides compositions and methods relatin to,g among other things, polypeptides that are capable of associating with nucleic acid molecules, wherein the polypeptides include at least one modified amino acid. In various embodiments, the polypeptides can be used for, e.g., delivering the nucleic acid molecules to a subject in need of gene therapy. Accordingly, the present disclosure includes, among other things, modified polypeptides capable of associating with nuclei acidc molecules, as well as compositions including modified polypeptides disclosed herein togethe withr associated nuclei acidc molecules. The present disclosure contemplate withouts, wishing to be bound by any particul ar scientif theoryic ,that association of a nucleic acid molecule with a modified polypeptide disclosed herein can facilita tedelivery of the nucleic acid to a target cell, subject, or other system. id="p-4" id="p-4" id="p-4" id="p-4" id="p-4"

[0004] In particular the, present disclosure includes, among other things, "modified mini - nucleosome core proteins" for delivery of nucleic acids. In various embodiments, a mini 1 nucleosome core protein of the present disclosure can include (a) a nucleic acid bindin gdomain ("NABD"); (b) a nuclei acidc releas edomain; and, optionally (c), furthe domainsr including, e.g., one or more of a targeting domain, a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, a mini-nucleosome core protein of the present disclosur cane include (a) a nucleic acid bindin gdomain ("NABD"); (b) a nucleic acid release domain; (c) a targeti ngdomain; and, optionally, (d) further domains including, e.g., one or more of a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, a mini-nucleosom coree protein of the present disclosure can be a modified mini - nucleosome core protein that includes (a) a nuclei acidc bindin gdomain ("NABD"); (b) a nucleic acid releas edomain; (c) a modified amino acid residue and,; optionally, (d) further domains including, e.g., one or more of a targeti ngdomain, a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, a mini-nucleosom coree protein of the present disclosure can be a modified mini-nucleosome core protein that includes (a) a nucleic acid bindin gdomain ("NABD"); (b) a nucleic acid release domain; (c) a targeting domain; (d) a modified amino acid residue and,; optionally, (e) further domains including, e.g., one or more of a stability domain, an oligomerization domain, and/or a linker domain. As disclosed in herein, a modified a mini-nucleosome core protein can be referred to and/or described as a mini- nucleosome core protein and/or as a protein that is or includes a mini-nucleosome core protein having at least one modified amino acid residue. Accordingl y,for clarity, all references, express or implie d,to "mini-nucleosom coree proteins" in the present disclosure include and encompass modified mini-nucleosom coree proteins of the present disclosure. One or more mini- nucleosome core proteins associated with a nucleic acid cargo can be referred to as a "loaded mini-nucleosome." Because a loaded mini-nucleosome that is for delivery of a nucleic acid to a target is non-viral, a mini-nucleosom is ean example of a non-viral vehicl fore nucleic acid delivery. id="p-5" id="p-5" id="p-5" id="p-5" id="p-5"

[0005] In particular the, present disclosure includes, among other things, "modified mini - nucleosome core proteins" for delivery of nucleic acids. In various embodiments, a mini- nucleosome core protein of the present disclosure can include (a) a nucleic acid bindin gdomain ("NABD"); (b) a targeting domain; and, optionally, (c) further domains including, e.g., one or more of a nucleic acid release domain, a stability domain, an oligomerization domain, and/or a 2 linker domain. In various embodiments, a mini-nucleosome core protein of the present disclosur cane include (a) a nucleic acid bindin gdomain ("NABD"); (b) a targeting domain; (c) a nucleic acid release domain; and, optionally, (d) further domains including, e.g., one or more of a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, a mini-nucleosome core protein of the present disclosure can be a modified mini-nucleosome core protein that includes (a) a nucleic acid bindin gdomain ("NABD"); (b) a targeting domain; (c) a modified amino acid residue and,; optionally, (d) further domains including, e.g., one or more of a nucleic acid release domain, a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, a mini-nucleosom coree protein of the present disclosure can be a modified mini-nucleosome core protein that includes (a) a nucleic acid bindin gdomain ("NABD"); (b) a targeting domain; (c) a nucleic acid release domain; (d) a modified amino acid residue and,; optionally, (e) further domains including, e.g., one or more of a stability domain, an oligomerization domain, and/or a linker domain. As disclosed in herein, a modified a mini- nucleosome core protein can be referred to and/or described as a mini-nucleosome core protein and/or as a protein that is or includes a mini-nucleosome core protein having at least one modified amino acid residue. Accordingl y,for clarity, all references, express or implied, to "mini-nucleosom coree proteins" in the present disclosure include and encompas smodified mini-nucleosom coree proteins of the present disclosure. One or more mini-nucleosome core proteins associated with a nucleic acid cargo can be referred to as a "loaded mini-nucleosome." Because a loaded mini-nucleosome that is for deliver ofy a nucleic acid to a target is non-vira l,a mini-nucleosom is ean example of a non-viral vehicle for nuclei acidc delivery. id="p-6" id="p-6" id="p-6" id="p-6" id="p-6"

[0006] The present disclosure includes the recognition that at least some modified mini - nucleosome core proteins of the present disclosure have advantageou propers ties including, without limitation, increased bioavailability; increased half-life increas; ed stability; decreased degradation; increased binding affinit e.g.,y, for target cells; increased uptake, e.g., by target cells; improved blood-brain barrier penetrati on;reduce daccumulatio e.g.,n, in target cells or tissue s;reduced aggregation; reduced precipitation; reduced therma denaturatl ion; and/or reduced kinetic denaturation, e.g., as set forth herein. 3 id="p-7" id="p-7" id="p-7" id="p-7" id="p-7"

[0007] The present disclosure includes the recognition that at least certain compositions and methods described herein remedy one or more deficiencies associated with AAV vectors, including that: id="p-8" id="p-8" id="p-8" id="p-8" id="p-8"

[0008] 1) AAV is associated with a payload limitation of 4.5 kb DNA lengt whichh, limitation prevents use of AAV in treatment of diseases caused at least in part by deficiency in expression of a gene product typically encoded by a nucleic acid larger than 4kb (for example genes like CFTR, HTT, F8, DMD, ABCA4 etc. cannot fit into AAV vectors) (Lai Y. et al, 2010). id="p-9" id="p-9" id="p-9" id="p-9" id="p-9"

[0009] 2) AAV has been known to integra atte low percentage and/or in a site-non- specific manner (Smith R.H., 2008). Random or site-non-specific integration may be deleterious if integration can or does disrupt a tumor suppressor gene or gene important for cellular functions. id="p-10" id="p-10" id="p-10" id="p-10" id="p-10"

[0010] 3) Depending on the serotype of AAV, 25-70 % of humans have preexisting neutralizing antibodies to AAV which means, they would be less likely to benefit for AAV therapy (Fitzpatrick Z., et al 2018). id="p-11" id="p-11" id="p-11" id="p-11" id="p-11"

[0011] 4) Multiple treatments with AAV are highly unlikely to be effective because once a patient is injected, the patient produce sa high numbe ofr antibodi esagainst the virus. For some diseases where cellul turnoverar is high (e.g., in the turnover of liver cells or airway epithel ial cells) multiple treatments maybe needed Thus,. due to increased antibodi esagainst AAVs following a first treatme nt,the same vecto mayr not be useful in follow-up treatments or doses. id="p-12" id="p-12" id="p-12" id="p-12" id="p-12"

[0012] 5) Effective treatment of some diseases may require deliver ofy an enormous payload of particles administered by intravenous injection in order to transduce cells in vivo. A high dose of AAV comes with its own toxicities which, are well documente (Hindd ere C.r et al, 2018). id="p-13" id="p-13" id="p-13" id="p-13" id="p-13"

[0013] 6) Most diseases are also associated with multi-organ defec tsand AAV may not be applied to various organs in the same body. One application at one site will raise antibodies and thus may block transducti aton other locations in the body when injected in a subseque nt treatment or dose. id="p-14" id="p-14" id="p-14" id="p-14" id="p-14"

[0014] Due at least in part to the deficiencies of AAV discusse dabove, there is a dire need for alternatives to AAV. In at least certain embodiments, non-viral vector discloss ed herein overcome one or more of the deficiencies of AAV discusse dabove. 4 id="p-15" id="p-15" id="p-15" id="p-15" id="p-15"

[0015] Moreover ,prior non-viral vectors are also associated with several barriers to therapeutic efficacy including: i) low transfection/transduction efficiency (Guerra-Crespo M et al, 2003) ii) low particle stability in blood, body fluids and other tissues (Barua, and Mitragotri, 2014); iii) low cell entry via receptor-mediated endocytosis or cell fusion; iv) low stability in, and low' escape from, endosomal and lysosomal compartment v)s, low' diffusion rate in the cytoplasm vi); low7 nuclea porer transit and; vii) low7 releas eof DNA to permi tbiological function in the nucleus (Zabner J et al, 1995). Several publications have documente inabilityd or low7 efficiency of prior non-viral vector tos transfect post-mitotic cells (Wilke M. et al, 1996).

Certain prior non-viral vectors lack longevity of expression and/or produce low amount of proteins that are not therapeutic enough and cannot be targeted to specific cell types in an efficie ntmanner. id="p-16" id="p-16" id="p-16" id="p-16" id="p-16"

[0016] Thus, despite state-of-the -arreset arch in the field of non-viral vector s,many prior non-viral vector ares not optimal for clinical use. Certai ncharacteristi csof at least certain embodiments discussed herein that contribute to, among other things, clinical utility, can include, without limitation: id="p-17" id="p-17" id="p-17" id="p-17" id="p-17"

[0017] Size and molecular weight: Many prior non-viral vectors that carry DNA molecule have a size of 10- 200nm in diameter (Konstan M.W. et. al, 2004). Their molecular weights can be greater than 300 kDa or greater than 500kDa. The present disclosure provides , among other things non-vir, al proteinaceou vehicles,s and/or loaded mini-nucleosomes that, are <20nm in diamet erand have a molecular weight of <500kDa. In particul arembodiments a non-, viral proteinaceous vehicles and/or, loaded mini-nucleosomes disclosed, herein can pass into the nucleus more efficiently, perhaps, by passive diffusion, at least in part because a typical nuclear pore is only 20nm in diameter, such that <20nm size may allow passage. id="p-18" id="p-18" id="p-18" id="p-18" id="p-18"

[0018] Stability in body fluids Many: prior non-viral vector ares degraded in body fluids like blood or CSF before they can be deliver edto target cells (Barua, and Mitragotr 2014).i, The present disclosur e,provides, among other things, non-viral proteinaceous vehicles and/or, loaded mini-nucleosomes, that are physiologically stable and/or have properties that allow them to be stable in blood and/or other body fluids until and after entry into a. target cell. At least one goal for these particles to safely reach the nucleus of desire dcells. id="p-19" id="p-19" id="p-19" id="p-19" id="p-19"

[0019] Release of particle ins nucleus: Many prior non-viral vector haves a very short life time because most releas eassociated nucleic acids before entering target cells, and. the remainder releas eassociated nucleic acids in the cytoplasm, where deliver edDNA encounte nucleasesrs that destroys DNA (Zabner ,J. et al, 1995). Certain prior vector thats make it into the cell nucleu s and provide expression levels are very low, if they express at all. The present disclosur alsoe recognizes, among other things, that it can be beneficial to releas eassociated nucleic acids at a slow rate, instead of all at once, which may allow for longevity of expression. id="p-20" id="p-20" id="p-20" id="p-20" id="p-20"

[0020] Cell type specificity: Prior non-viral vector ares not targeted to specific cell types are associated with reduced level ofs transduction and thus, reduced expression. The present disclosur providese among, other things, non-viral vector optimizs ed for cell-type specificit y.

Certain means of engineering cell-typ specife icit arey described, e.g., in Templeton and. Senzer , 2011. id="p-21" id="p-21" id="p-21" id="p-21" id="p-21"

[0021] Taken together, there is a tremendous need for nucleic acid, deliver technologiesy that provide effective levels of expression for a desire dduration, are non-immunogenic and non- toxic, and have less limited payload capacity. Moreover, the need for millions of patients of Huntington, Stargardt, Duchenne muscul ardystrophy, Cystic Fibrosis, and other conditions treatable by gene therapy clearly presents a need for technology that can help trea theset patients. id="p-22" id="p-22" id="p-22" id="p-22" id="p-22"

[0022] The present disclosure provides safe and efficacious non-viral proteinaceous vehicles ("mini-nucleosom coree proteins"), and loaded mini-nucleosomes for, deliver ofy nucleic acids. The present disclosure further recognizes that in at least some instances modified mini-nucleosom coree proteins of the present disclosure can have advantageou propers ties including, without limitati on,increased bioavailability; increased half-life; increased stability; decrease ddegradation; increased binding affinit e.g.,y, for target cells; increased uptake, e.g., by target cells; improve dblood-brai nbarrier penetrati on;reduced accumulatio e.g.,n, in target cells or tissue s;reduced aggregation; reduced precipitation; reduced thermal denaturation; and/or reduced kinetic denaturation, e.g., as set forth herein. id="p-23" id="p-23" id="p-23" id="p-23" id="p-23"

[0023] In various embodiments, a mini-nucleosom coree protein is associated with one or more nuclei acids.c As disclosed herein a mini-nucleosome core protein associated with one or more nucleic acids can be referred, to as a "loaded mini-nucleosome." 6 id="p-24" id="p-24" id="p-24" id="p-24" id="p-24"

[0024] In various embodiments, a mini-nucleosom coree protein includes a nucleic acid releas edomain that targets a loaded mini-nucleosom to eone or more specifi ccell types for delivery and/or targeted expression of a nuclei acid,c such as a gene, in or to one or more specific cell types. id="p-25" id="p-25" id="p-25" id="p-25" id="p-25"

[0025] In various embodiments, a mini-nucleosom coree protein composition (e.g., a composition including one or more loaded mini-nucleosom canes) be titered and/or administered either once or repeatedly based on need. Furthermore in, various embodiments, a mini- nucleosome core protein or mini-nucleosom compositie on (e.g., a composition including one or more loaded mini-nucleosomes) is non-immunogenic and non-toxic. id="p-26" id="p-26" id="p-26" id="p-26" id="p-26"

[0026] Mini-nucleosome core proteins disclosed herein can, in certain embodiments, utilize principles applicable to macromolecule uptake, viral entry into cells, nucleosom e formation in eukaryotic cell s,cleavage of certain proteins at certain location in the cells, etc. id="p-27" id="p-27" id="p-27" id="p-27" id="p-27"

[0027] Various embodiments of the compositions and methods provided herein include domains that facilita teone or more of enhanced stability, targeting to specific cell types, and enhanced longevi ofty expression by slow nucleic acid release. id="p-28" id="p-28" id="p-28" id="p-28" id="p-28"

[0028] In various embodiments, a mini-nucleosom coree protein and/or a mini- nucleosome is stabl ein body fluids and/or include domains that allow and/or target releas ein or to the nucleus. In at least a first aspect, the present disclosure provides an engineered polypeptid e including a nucleic acid binding domain and a nucleic acid release domain, where one or more amino acids of the engineered polypeptid ise a modified amino acid, optionall wherey the modification includes at least one of: (i) phosphorylation; (ii) sulfation; (iii) glycosylation (iv); prenylation; (v) methylation; (vi) sialylation; (vii) lipidation and/or lipoylation; (viii) acetylation; (ix) hydroxylation; (x) palmitoylation; (xi) mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylatio and/orn; (xv) any combination thereof. In certain embodiments, the engineered polypeptide includes a targeting domain. id="p-29" id="p-29" id="p-29" id="p-29" id="p-29"

[0029] In at least a further aspect, the present disclosure provide ans engineered polypeptide including a nucleic acid bindin gdomain and a targeting domain, where one or more amino acids of the engineered polypeptid ise a modified amino acid, optionall wherey the modification includes at least one of: (i) phosphorylation; (ii) sulfation; (iii) glycosylation (iv); prenylation; (v) methylation; (vi) sialylation; (vii) lipidation and/or lipoylation; (viii) acetylation; 7 (ix) hydroxylation; (x) palmitoylation; (xi) mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylatio and/orn; (xv) any combination thereof; where engineered polypeptide optionall furthey includesr a nucleic acid releas edomain. id="p-30" id="p-30" id="p-30" id="p-30" id="p-30"

[0030] In at least certain embodiments of various aspects of the present disclosure, each of two or more amino acids of an engineered polypeptide is a modified amino acid. In some embodiments, at least one of the modified amino acids includes a modification chain including two or more modifications selected from: (i) phosphorylation; (ii) sulfation; (iii) glycosylation; (iv) prenylation; (v) methylation; (vi) sialylation; (vii) lipidation and/or lipoylation; (viii) acetylatio (ix)n; hydroxylation; (x) palmitoylation (xi); mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylatio andn; (xv) any combination thereof. In some embodiments, the modification increases the stability, half-life, and/or bioavailabilit ofy the engineered polypeptid e.In some embodiments, the modification increases the affinity and/or avidity of the engineered polypeptide with a binding partner optionall, wherey the binding partner is a receptor , cell, or cell membrane .In some embodiments, the modification increases the affinity or avidity of the engineered polypeptide with a nuclei acid.c In some embodiments, the modification decreases precipitation and/or aggregation of the engineered polypeptide. id="p-31" id="p-31" id="p-31" id="p-31" id="p-31"

[0031] In at least certain embodiments of various aspects of the present disclosure, a nucleic acid binding domain is derived from a histone polypeptide sequence. In some embodiments, the nucleic acid bindin gdomain is or includes the amino acid sequenc KRHRKe .

In some embodiments, the nucleic acid binding domain is or includes an amino acid sequence that includes KRHRK, RRRRR, RRLARR, KKAKAAAKPKK, KKDGKKRKR, KKKLK, KKRIRK, RKKSK, KKPKK, or a combination thereof In. some embodiments, the nucleic acid bindin gdomain is a modified nucleic acid binding domain in that the nucleic acid binding domain includes one or more modified amino acids. id="p-32" id="p-32" id="p-32" id="p-32" id="p-32"

[0032] In at least certain embodiments of various aspects of the present disclosure, a targeting domain is a targeti ngdomain having the sequenc ofe any one of SEQ ID NOs: 397- 422, where the targeti ngdomain is phosphorylated. In at least certain embodiments of various aspects of the present disclosur e,a targeti ngdomain is a targeting domain having the sequenc ofe any one of SEQ ID NOs: 423-428, where the targeti ngdomain is sulfated. In at least certain embodiments of various aspects of the present disclosure, a targeting domain is a targeting 8 domain having the sequenc ofe any one of SEQ ID NOs: 429-434, where the targeting domain is glycosylated. id="p-33" id="p-33" id="p-33" id="p-33" id="p-33"

[0033] In at least certain embodiments of various aspects of the present disclosure, a targeting domain is a targeti ngdomain having the sequenc ofe any one of SEQ ID NOs: 435- 440, where the targeti ngdomain is prenylated. In at least certain embodiments of various aspects of the present disclosur e,a targeti ngdomain is a targeting domain having the sequenc ofe any one of SEQ ID NOs: 441-446, where the targeti ngdomain is methylated. In at least certain embodiments of various aspects of the present disclosure, a targeting domain is a targeting domain having the sequenc ofe any one of SEQ ID NOs: 447-459, where the targeting domain is sialylated. In at least certain embodiment ofs various aspects of the present disclosur e,a targeting domain is a cell attachment targeting domain, a beta galactos bindie ng domain, a fucose bindin gdomain, a heparin binding domain, a sialic acid bindin gdomain, a glycoprote bindingin domain, a carbohydrate binding domain, a lysophosphatidic acid bindin gdomain, a cAMP bindin gdomain, a hyaluronan binding domain, a chondroitin sulfate bindin gdomain, an integrin bindin gdomain, a nucleolin binding domain, a collagen bindin gdomain, a clathrin binding domain, a Fc receptor binding domain, an actin binding domain, an endocytosis motif, a nuclear localization signal, or a combination thereof. id="p-34" id="p-34" id="p-34" id="p-34" id="p-34"

[0034] In at least certain embodiments of various aspects of the present disclosure, a targeting domain is a cell attachment targeting domain. In some embodiments, the cell attachment targeting domain is or includes an amino acid sequenc thate includes WGREERQ, NTQIH, WNNKTPH, TPH, VNRWS, XBBBXXBX, ARKKAAKA, QRR, SRR, WEPSRPFPVD, HRRTRKAPKRIRLPHIR, KRTGQYKLGSKTGPGQK, KKTK, KLRSQLVKK, RRRCGQKKK, BX(7)B, RIQNLLKITNLRIKFVK, KKEKDIMKKTI, KGE, RGD, RGDS, TTVVNPKYEGK, ERMSQIKRLLS, WRHRARS, GFOGER, LFDLM, WGREERQ, QSTEKRG, LPNTG, or a combination thereof. id="p-35" id="p-35" id="p-35" id="p-35" id="p-35"

[0035] In at least certain embodiments of various aspects of the present disclosure, a targeting domain is an internalization domain. In some embodiments, the internalization domain is or includes an amino acid sequenc thate includes FXDXF, PPSY, FEDNFVP, YIRV, YADW, YTQV, KKRPKP, SSDDE, RRASS, (YXXL)2, LPLTG, LAFTG, or a combination thereof. 9 id="p-36" id="p-36" id="p-36" id="p-36" id="p-36"

[0036] In at least certain embodiments of various aspects of the present disclosure, a targeting domain is a cell-typ specie fic targeti ngdomain. In some embodiments, the cell-type specific targeti ngdomain is or includes an amino acid sequenc thate includes ASSLNIA, KKEEEKKEEEKKEEE, LIFHKEQ, KFNKPFVFLI, QPEHSST, EYHHYNK, NGR, GEKGEP, KTKKK, KALKKK, KGKKK, CSVTCG, ERE, YKYNLNGRES, YRSL, KGGK7, KKKQYTSIHHG, KDEL, LADQDYTKTA, or a combination thereof. id="p-37" id="p-37" id="p-37" id="p-37" id="p-37"

[0037] In at least certain embodiments of various aspects of the present disclosure, a targeting domain is a modified targeting domain in that the targeting domain includes one or more modified amino acids. In some embodiments, the nuclei acidc releas edomain is or includes an amino acid sequence that includes GRKKRRQRRRPQ. KRH, KSVKKRSVSEIQ, NRRKKRAL, KFERQ, VRGP, NKDS, NRDN, ANNR, or a combination thereof. id="p-38" id="p-38" id="p-38" id="p-38" id="p-38"

[0038] In at least certain embodiments of various aspects of the present disclosure, a the nucleic acid release domain is a modified nucleic acid release domain in that the nucleic acid releas edomain includes one or more modified amino acids. id="p-39" id="p-39" id="p-39" id="p-39" id="p-39"

[0039] In at least certain embodiments of various aspects of the present disclosure, an engineered polypeptide further includes a poly-arginine domain. In some embodiments, the poly-arginine domain is a modified poly-arginine domain in that the poly-arginine domain includes one or more modified amino acids. id="p-40" id="p-40" id="p-40" id="p-40" id="p-40"

[0040] In at least certa ni embodiments of various aspects of the present disclosure, an engineered polypeptide further includes a nuclear internalization signal or a nuclear import machinery binding domain. In some embodiments, the nuclear internalization signal or nuclear import machinery binding domain is or includes an amino acid sequenc thate includes KKK YKLK. KKRKLE, TRSK, HRKRKR, NKRKRK, AEKSKKK, RKSK, KRVK, KRK, LQQTPLHLAVI, RRPR, PRPR, RPPP. RKKRKGK, PA AKR VK LD, Ki .K ؛ KRPVK.

PKKKRKV, QRKRQK, DSPE, FQVT, QSTEKRG, RQGLID, Cyclic RKKH, or a. combination thereof. In some embodiments the, nuclear internalization signal or a nuclear import machinery bindin gdomain is a modified nuclear internalization signal or a nuclea imporr t machinery bindin gdomain in that the nuclear internalization signal or a nuclea imporr t machinery binding domain includes one or more modified amino acids. id="p-41" id="p-41" id="p-41" id="p-41" id="p-41"

[0041] In at least certain embodiments of various aspects of the present disclosure, an engineered polypeptide further includes a stability domain. In some embodiments, the stability domain is or includes an amino acid sequenc thate includes YTRF, GDAY, LLEE, RKKRRQRRR, YKSL, YENF, FQDL, YIGSR, IKVAV, or a combination thereof In. some embodiments, the stability domain is a modified stability domain in that the stability domain includes one or more modified amino acids. id="p-42" id="p-42" id="p-42" id="p-42" id="p-42"

[0042] In at least certain embodiments of various aspects of the present disclosure, an engineered polypeptide further includes an oligomerization domain. In some embodiments, the oligomerization domain is selected from the oligomerization domains of Table 11, optionally where the oligomerization domain is positioned at the C-terminus of the engineered polypeptide.

In some embodiments, the oligomerization domain is a modified oligomerization domain in that the oligomerization domain includes one or more modified amino acids. id="p-43" id="p-43" id="p-43" id="p-43" id="p-43"

[0043] In at least certain embodiments of various aspects of the present disclosure, an engineered polypeptide further includes a linker. In some embodiments, the linker is a linker according to any one of SEQ ID NOs: 154-250. In some embodiments, the linker is a modified linker in that the linker includes one or more modified amino acids. id="p-44" id="p-44" id="p-44" id="p-44" id="p-44"

[0044] In some embodiments, one or more amino acids of an engineered polypeptide is a phosphorylated amino acid. In some embodiments, the phosphorylated amino acid is a serine, threonine, or tyrosine amino acid. In some embodiments, the phosphorylated amino acid is present in a linker domain or targeti ngdomain. id="p-45" id="p-45" id="p-45" id="p-45" id="p-45"

[0045] In some embodiments, one or more amino acids of an engineered polypeptide is a sulfate aminod acid. In some embodiments, the sulfat edamino acid is a serine, threonin e,or tyrosine amino acid. In some embodiments, the sulfate aminod acid is present in a linker domain or targeti ngdomain. id="p-46" id="p-46" id="p-46" id="p-46" id="p-46"

[0046] In some embodiments, one or more amino acids of an engineered polypeptide is an acetylated amino acid. In some embodiments, the acetylated amino acid is a lysine amino acid. In some embodiments, the acetylated amino acid is present in a linker domain or targeting domain. id="p-47" id="p-47" id="p-47" id="p-47" id="p-47"

[0047] In some embodiments, one or more amino acids of an engineered polypeptide is a mannosylat edamino acid. In some embodiments, the mannosylated amino acid is a serin eamino 11 acid. In some embodiments, the mannosylat aminoed acid is present in a linker domain or targeting domain. id="p-48" id="p-48" id="p-48" id="p-48" id="p-48"

[0048] In at least one further aspect, the present disclosure provide as polynucleotide that encodes the amino acid sequenc ofe an engineered polypeptide of the present disclosure, such as a DNA or RNA polynucleotide. In at least one further aspect ,the present disclosure provides a vector including polynucleotide of the present disclosure. In at least one further aspect, the present disclosure provides a cell including an engineered polypeptide of the present disclosure or a vector of the present disclosure. id="p-49" id="p-49" id="p-49" id="p-49" id="p-49"

[0049] In at least one further aspect, the present disclosure provide methods of making an engineered polypeptide of the present disclosure, the method including expressing a polynucleotide of the present disclosure in a cell. In some embodiments the, method further includes isolating the engineered polypeptide from the cell. id="p-50" id="p-50" id="p-50" id="p-50" id="p-50"

[0050] In at least one further aspect, the present disclosure provide as composition including: (i) at least one polynucleotide and ,(ii) at least one engineered polypeptide of the present disclosure. In some embodiments, the at least one polynucleotide is or includes DNA or RNA. In some embodiments, the at least one polynucleotide includes a nucleotide sequence encoding a polypeptid e.In some embodiments the, at least one polynucleotide is or includes mRNA. In some embodiments, the at least one polynucleotide includes an inhibitory RNA. In some embodiments, the inhibitory RNA is a gRNA, siRNA, miRNA, or shRNA. In some embodimen thet, composition includes at least two engineered polypeptides of the present disclosure, where a first engineered polypeptide of present disclosure is able to oligomeriz withe a second engineered polypeptide of the present disclosure. In some embodiments, the ratio of polynucleotides to engineered polypeptide is sbetween 1:1 and 1:2,000. In some embodiments, the ratio of polynucleotides to engineered polypeptides is between 1:1 and 1:1,000, between 1:1 and 1:500, between 1:1 and 1:200, between 1:1 and 1: 100, between 1:1 and 1:50, between 1:3 and 1:1,000, between 1:3 and 1:500, between 1:3 and 1:200, between 1:3 and 1: 100, or between 1:3 and 1:50. In some embodiments, the ratio of polynucleotides to engineered polypeptides is between 1:200 and 1:2,000, between 1:200 and 1:1000, or between 1:200 and 1:500. In some embodiments, the composition includes a pharmaceutical carrier. 12 id="p-51" id="p-51" id="p-51" id="p-51" id="p-51"

[0051] In at least one further aspect, the present disclosure provide as method that includes administering a composition of the present disclosure to a system, where the system is a cell, tissue, or subject. In some embodiments afte, administr rati on,the modification increases the stability, half-life, and/or bioavailabili ofty the composition in the system. In some embodiments, after administratio then, modification increases the affinit ory avidity of the composition with a bindin partnerg in the system, optionall wherey the bindin gpartner is a receptor cell,, or cell membrane .In some embodiments, after administrati on,the modification decreases precipitation and/or aggregation of the composition in the system. In some embodiments, after administratio then, modification increases the rate at which the composition enter ones or more cells in the system. In some embodiments, afte adminisr tration, the modification increases deliver ofy the composition to one or more cells in the system. In some embodiments, after administratio then, modification increases delivery of the nucleic acid of the composition to one or more cells in the system. In some embodiments, the system is a mammalian subjec tand, after administrati on,the modification decreases accumulation of the composition in liver. In some embodiments, the system is a mammalian subjec tand, after administrati on,the modification increases the amount of composition that crosses the blood- brain barrier. id="p-52" id="p-52" id="p-52" id="p-52" id="p-52"

[0052] In some embodiments, the present disclosure provides a method that includes administering a composition of the present disclosure including at least one polynucleotide and at least one engineered polypeptide to a cell, tissue, or subject where, in one or more amino acids of the engineered polypeptide is a phosphorylated amino acid. In some embodiments, the phosphorylated amino acid is a serine, threonine, or tyrosine amino acid. In some embodiments, the phosphorylated amino acid is present in a linker domain or targeting domain. In some embodiments, the composition is deliver edto cells of the centr alnervou systems (CNS). id="p-53" id="p-53" id="p-53" id="p-53" id="p-53"

[0053] In some embodiments, the composition is delivere tod CNS neurons. In some embodiments, the composition is deliver edto CNS astrocytes, microglia, oligodendrocytes, or glia. In some embodiments, the composition is delivered to spinal cord cell s,optionall wherey in the spina cord cells are spinal cord neuron ors spinal cord glial cell s.In some embodiments, the polynucleoti encodesde an expression product that is expressed in cells to which the composition 13 is delivered. In some embodiments the, subject is a mammalia nsubjec tand the administration is intrathecal, intracranial or, intra-cistema magna. id="p-54" id="p-54" id="p-54" id="p-54" id="p-54"

[0054] In some embodiments, the present disclosure provides a method that includes administering a composition of the present disclosure including at least one polynucleotide and at least one engineered polypeptide to a cell, tissue, or subject where, in one or more amino acids of the engineered polypeptide is a sulfate aminod acid. In some embodiments, the sulfated amino acid is a serine, threonin e,or tyrosine amino acid. In some embodiments, the sulfate d amino acid is present in a linker domain or targeti ngdomain. In some embodiments, the composition is delivered to cells of the centr alnervous system (CNS). In some embodiments, the composition is deliver edto CNS neurons. In some embodiments, the composition is deliver edto CNS astrocytes, microglia, oligodendrocyt ores, glia. In some embodiments, the composition is deliver edto spinal cord cell s,optionall wherey in the spina cord cells are spinal cord neurons or spinal cord glial cells. In some embodiments the, polynucleotide encodes an expression product that is expressed in cells to which the composition is delivere d.In some embodiments, the subject is a mammalian subject and the administration is intratheca l, intracranial or, intra-cisterna magna. id="p-55" id="p-55" id="p-55" id="p-55" id="p-55"

[0055] In some embodiments, the present disclosure provides a method that includes administering a composition of the present disclosure including at least one polynucleotide and at least one engineered polypeptide o a cell, tissue, or subject, wherein one or more amino acids of the engineered polypeptide is an acetylated amino acid. In some embodiments, the acetylated amino acid is a lysine amino acid. In some embodiments, the acetylated amino acid is present in a linker domain or targeting domain. In some embodiments, the composition is deliver edto CNS neurons. In some embodiments, the composition is deliver edto retinal cells. In some embodiments, the composition is delivered to retinal neurons, optionall whery ein the retinal neurons include one or more of photoreceptor bipolars, cells, retinal ganglion cell s,horizontal cells, and amacrine cell s.In some embodiments, the composition is deliver edto photoreceptor cells, optionall whery ein the photoreceptor cells include one or both of rods and cones. In some embodiments, the polynucleotide encodes an expression product that is expressed in cells to which the composition is delivere d.In some embodiments, the subject is a mammalian subjec t and the administrat ionis intravitre supracal, horoidal, or subretinal. 14 id="p-56" id="p-56" id="p-56" id="p-56" id="p-56"

[0056] In some embodiments, the present disclosure provides a method that includes administering a composition of the present disclosure including at least one polynucleotide and at least one engineered polypeptide to a cell, tissue, or subject where, in one or more amino acids of the engineered polypeptide is a mannosylated amino acid. In some embodiments, the mannosylat edamino acid is a serine amino acid. In some embodiments the, mannosylated amino acid is present in a linker domain or targeting domain. In some embodiments, the composition is deliver edto CNS neurons. In some embodiments, the composition is deliver edto retinal cells .

In some embodiments, the composition is deliver edto retinal neurons, optionall wherey in the retinal neurons include one or more of photoreceptor bipolars, cell s,retinal ganglion cell s, horizontal cells, and amacrine cell s.In some embodiments, the composition is delivered to photoreceptor cells include one or both of rods and cones. In some embodiments, the polynucleotide encodes an expression product that is expressed in cells to which the composition is delivered. In some embodiments the, subject is a mammalian subject and the administration is intravitre suprachoroidal,al, or subretinal. id="p-57" id="p-57" id="p-57" id="p-57" id="p-57"

[0057] In at least one further aspect, the present disclosure provide as method of condensing a polynucleot ide,including contacting the polynucleotide with a polypeptide of the present disclosure. id="p-58" id="p-58" id="p-58" id="p-58" id="p-58"

[0058] In at least one further aspect, the present disclosure provide as method of neutralizing the charge of a polynucleot ide,including contacting the polynucleotide with a polypeptide of the present disclosure. id="p-59" id="p-59" id="p-59" id="p-59" id="p-59"

[0059] In at least one further aspect, the present disclosure provide as composition including an engineered polypeptide and at least one nuclei acid,c the engineered polypeptid e including a nucleic acid binding domain, a targeti ngdomain, and a nuclei acidc releas edomain, where one or more amino acids of the engineered polypeptide is a modified amino acid, optionall wherey the modification includes at least one of: (i) phosphorylation; (ii) sulfation; (iii) glycosylation (iv); prenylation; (v) methylation; (vi) sialylation; (vii) lipidation and/or lipoylation; (viii) acetylation; (ix) hydroxylation; (x) palmitoylation; (xi) mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylatio and/orn; (xv) any combination thereof ; optionall wherey the composition is for use in delivering a nucleic acid to a subject or system. id="p-60" id="p-60" id="p-60" id="p-60" id="p-60"

[0060] In at least one further aspect, the present disclosure provide as composition including an engineered polypeptide and at least one nuclei acid,c the engineered polypeptid e including a nucleic acid binding domain and a targeting domain, where one or more amino acids of the engineered polypeptide is a modified amino acid, optionall wherey the modification includes at least one of: (i) phosphorylation; (ii) sulfation; (iii) glycosylation; (iv) prenylation; (v) methylation; (vi) sialylation; (vii) lipidation and/or lipoylation; (viii) acetylatio (ix)n; hydroxylation; (x) palmitoylation (xi); mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylatio and/orn; (xv) any combination thereof; optional lywhere the composition is for use in delivering a nucleic acid to a subject or system. In some embodiments the, engineered polypeptide comprises a targeting domain. id="p-61" id="p-61" id="p-61" id="p-61" id="p-61"

[0061] In at least one aspect ,the present disclosure provides an engineered polypeptid e that includes a nuclei acidc binding domain and a. targeting domain, which engineered polypeptide can be a mini-nucleosom coree protein. A loaded mini-nucleosome can be or provide a non-viral vector that includes an engineered polypeptide (e.g., a mini-nucleosom coree protein) as described herein and at least one nucleic acid molecul ase provided herein or otherwise known in the art. id="p-62" id="p-62" id="p-62" id="p-62" id="p-62"

[0062] In some embodiments, an engineered polypeptide (e.g., a mini-nucleosom coree protein) that is or includes a nucleic acid binding domain is derived from a histone polypeptide sequenc and/ore a nucleic acid binding domain that is or includes the amino acid sequence KRHRK. In certain embodiments, an engineered polypeptide of the present disclosure includes a. nucleic acid binding domain that is or includes an amino acid sequenc thate includes KRHRK, RRRRR, RRLARR, KKAKAAAKPKK, KKDGKKRKR, KKKLK, KKRIRK, RKKSK, KKPKK, or a. combination thereof, but not limited to it. id="p-63" id="p-63" id="p-63" id="p-63" id="p-63"

[0063] In some embodiments, an engineered polypeptide of the present disclosure includes a nucleic acid bindin gdomain derived from any histone protein sequenc orthosee described in Table 3 or a combination of the sequences described herein but not limited to it.

These nucleic acid binding domains may be derived from various human proteins or other organisms. One skilled in the art. may contemplate modifying or engineering the "NABD" with changes to the amino acid, sequence. One skilled in the art may also contemplate placing the 16 "NABD" in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslationa modificationsl to amino acids. id="p-64" id="p-64" id="p-64" id="p-64" id="p-64"

[0064] In some embodiments, an engineered polypeptide of the present disclosure includes a targeti ngdomain that is a cell attachme nttargeting domain, a beta galactose binding domain, a fucos ebinding domain, a heparin bindin gdomain, a sialic acid binding domain, a. glycoprote bindinin gdomain, a carbohydrate binding domain, a lysophosphatidi acidc binding domain, a. cAMP binding domain, a hyaluronan bindin gdomain, a chondroitin sulfate binding domain, an integrin binding domain, a nucleohn binding domain, a collagen binding domain, a clathri nbindin gdomain, a Fc receptor binding domain, an actin binding domain, an endocytosis motif, a nuclea localizatir onsignal, or a. combination thereof but not limited to it. Some example s of those domain are described in Table 5 but is not limited to these. These domains may be derived from any human proteins or other organisms. One skilled in the art may contemplate modifying or engineering the targeting domain with changes to the amino acid, sequence. One skilled in the art may also contemplate placing the targeting domain in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslational modifications to amino acids. id="p-65" id="p-65" id="p-65" id="p-65" id="p-65"

[0065] In some embodiments, an engineered polypeptide of the present disclosure includes a targeti ngdomain that is an internalization domain wherein the internalization domain is or includes an amino acid sequenc thate includes FXDXF, PPSY, FEDNFVP, YIRV, YADW, YTQV, KKRPKP, SSDDE, RRASS, (YXXL)2, LPLTG, LAFTG, or a combination thereof but not limited to it. These domains may be derived from human proteins or other organisms. One skilled in the art may contemplate modifying or engineering the internalization domain with changes to the amino acid sequence. One skilled in the art may also contemplate placing the internalizatio domainn in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslational modifications to amino acids. id="p-66" id="p-66" id="p-66" id="p-66" id="p-66"

[0066] Those of skill in the art will appreciate that, as used in protein sequences throughout the present specification, an "X" can refer to any amino acid unles otherwiss e specified. Thus, unless otherwise specified, an "X" is a. placeholder for a. single amino acid, which position could be filled by any single amino acid known to those of skill in the art. 17 id="p-67" id="p-67" id="p-67" id="p-67" id="p-67"

[0067] In some embodiments, an engineered polypeptide of the present disclosure includes a cell attachment targeting domain that is or includes an amino acid sequenc selectede from WGREERQ, XTQM L WNNKTPH, TPH, VNRWS, XBBBXXBX, ARKKAAKA, QRR, SRR, WEPSRPFPVD, MRRTRKAPKRIRLPHIR, KRTGQYKLGSKTGPGQK, KKTK, KLRSQLVKK, RRRCGQKKK, BX(7)B, RIQNLLKITNLRIKFVK, KKEKDIMKKTI, KGE, RGD, RGDS, TTVVNPKYEGK, ERMSQIKRLLS, WRHRARS, GFOGER, LFDLM, WGREERQ, QSTEKRG, LPNTG, and a. combination thereof, where X can be any amino acid, but not limited to it. id="p-68" id="p-68" id="p-68" id="p-68" id="p-68"

[0068] In some embodiments, an engineered polypeptide of the present disclosure includes a. targeti ngdomain that is an internalizatio domainn cell-typ specifie ctargeting domain wherein the cell-type specific targeting domain is or includes an amino acid sequenc thate includes ASSLNIA, KKEEEKKEEEKKEEE, LIFHKEQ, KFNKPFVFLI, QPEHSST, EYHHYNK, NGR, GEKGEP, KTKKK, KALKKK, KGKKK, CSVTCG, ERE, YKYNLNGRES, YRSL, KGGK7, KKKQYTSIHHG, KDEL, LADQDYTKTA, or a combination thereof but not limited to it. These domains may be derived from human proteins or other organisms. One skilled in the art may contemplate modifying or engineering the targeting domain with changes to the amino acid sequence. One skilled in the art may also contempl ate placing the targeti ngdomain in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslationa modifil cations to amino acids. id="p-69" id="p-69" id="p-69" id="p-69" id="p-69"

[0069] In some embodiments, an engineer edpolypeptide of the present disclosure includes a poly-arginine domain with varying length or multiple polv-arginine domains throughout the polypeptid sequence.e id="p-70" id="p-70" id="p-70" id="p-70" id="p-70"

[0070] In some embodiments, an engineered polypeptide of the present disclosure includes a nuclear internalization signal or a nucle arimport machinery binding domain .The engineered polypeptide, the nuclear internalization signal or a nuclea imporr t machinery binding domain can be or include an amino acid sequence that includes KKKYKLK, KKRKLE, TRSK, HRKRKR, NKRKRK, AEKSKKK, RKSK, KRVK, KRK, LQQTPLHLAVI, RRPR, PRPR, RPPP, RKKRKGK, PAAKRVKLD, KLK1KRPVK, PKKKRKV, QRKRQK, DSPE, FQVT, QSTEKRG, RQGLID, Cyclic RKKH, or a combination thereof but not limited to it. These domains may be derived from human proteins or other organisms. One skilled in the art may 18 contemplate modifying or engineering the nuclear internalization signal with changes to the amino acid sequence. One skilled, in the art may also contempl ateplacing the nucle ar internalization signal in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslational modifications to amino acids. id="p-71" id="p-71" id="p-71" id="p-71" id="p-71"

[0071] In some embodiments, an engineered polypeptide of the present disclosure includes a nucleic acid release domain. The nucleic acid release domain is or includes an amino acid sequenc thate includes GRKKRRQRRRPQ, KRH, KSVKKRSVSEIQ, NRRKKRAL, KFERQ, VRGP, NKDS, NRDN, ANNR, or a combination thereof but not limited to it. These domains may be derive dfrom various proteins that are substrates of peptidases, enzymes or other proteins foun din humans or other organisms. Some nucleic acid release domains may also be derive dfrom autolys issites of various proteins. One skilled in the art may contempl ate modifying or engineering the nucleic acid releas edomain with changes to the amino acid sequence. One skilled in the art may also contemplate placing the nucleic acid release signal in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslational modifications to amino acids. id="p-72" id="p-72" id="p-72" id="p-72" id="p-72"

[0072] In some embodiments, an engineer edpolypeptide of the present disclosure further including a stability domain. In some embodiments, an engineered polypeptide of the present disclosur cane include a stability domain that is or includes an amino acid sequenc thate includes YTRT. GDAY, LLEE, RKKRRQRRR, YKSL, YENF, FQDL, YIGSR, IKVAV, or a combination thereof but not limited, to it. These domains may be derived from human proteins or other organisms. One skilled in the art. may contemplate modifying or engineering the stability domain with changes to the amino acid sequence. One skille ind the art may also contempl ate placing the stability domain in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslational modifications to amino acids. id="p-73" id="p-73" id="p-73" id="p-73" id="p-73"

[0073] In some embodiments, an engineer edpolypeptide of the present disclosure includes an oligomerization domain. In some embodiments, an engineered polypeptide of the present disclosure can include an oligomerization domain is selected from the oligomerization domains of Table 11 but. not limited to it. The position of oligomerization domain is positioned at the C-terminus of an engineered polypeptide of the present disclosure or at any other locations.

These domains may be derived from human proteins or other organisms. One skilled in the art. 19 may contemplate modifying or engineering the oligomerization domain with changes to the amino acid sequence. One skilled, in the art may also contempl ateplacing the oligomerization domain in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslationa modificationsl to amino acids. id="p-74" id="p-74" id="p-74" id="p-74" id="p-74"

[0074] In some embodiments, an engineered polypeptide of the present disclosure includes a Linker. In some embodiments, an engineered polypeptide of the present disclosure can include a. !..inker selected, without limitation, from the exemplary domains of Table 12. The position of linker in an engineered polypeptid ofe the present disclosure may be in betwee othern domains and any other locations. These Linkers may be derived from human proteins or other organisms. One skilled in the art. may contemplate modifying or engineering the linker domain with changes to the amino acid sequence. One skilled, in the art may also contemplate placing the linker domain in reverse sequenc ore by switching amino acid positions within the domain or adding posttranslatio modificationsnal to amino acids. id="p-75" id="p-75" id="p-75" id="p-75" id="p-75"

[0075] In various embodiments, two or more engineered polypeptides of the present disclosur cane oligomerize. id="p-76" id="p-76" id="p-76" id="p-76" id="p-76"

[0076] In some embodiments, the present disclosure includes a. composition that includ es an engineered polypeptide of the present disclosure (e.g., a mini-nucleosome core protein) togethe withr at least one polynucleot ide.In some embodiments, the polypeptide is a DNA or RNA polynucleot ide.In some embodiments, the polypeptide is a. or includes an inhibitory RNA, wherein the inhibitory' RNA is a gRNA, siRNA, miRNA, or shRNA. In various embodiments, the polypeptide(s) and polynucleotide are(s) not associated but. are together in a. composition, e.g., a kit or solution. In various embodiments, the polypeptide(s) and polynucleotide( ares) associated e.g.,, condensed, e.g., to form a loaded mini-nucleosome. In certai nembodiments, the ratio of polynucleotides to engineered polypeptides is between 1:1 and 1:2,000 or betwee n1:3 and 1:2,000. In certain embodiments, the ratio of polynucleotides to engineered polypeptides is between 1:1 and 1:2,000. In certain embodiments, the ratio of polynucleotides to engineered polypeptides is betwee n1:1 and 1:1,000, between 1:1 and 1:500, between 1:1 and 1:200, between 1:1 and I: 100, between 1:1 and 1:50. In certain embodiments, the ratio of polynucleotides to engineered polypeptide is sbetween 1:3 and 1: 1,000, between 1:3 and. 1:500, between 1:3 and 1:200, between 1:3 and 1: 100, or between 1:3 and 1:50. In certain embodiments, the ratio of polynucleot idesto engineered polypeptides is between 1:200 and 1:2,000, between 1:200 and 1:1000, or between 1:200 and 1:500. In certain embodiments, the ratio of polynucleotides to engineered poly peptides is between 1:1 and 1:50, 1:1 and 1:40, 1:1 and 1:30, 1:1 and. 1:20, 1:1 and 1:10, 1:1 and 1:5, 1:1 and. 1:4, 1:1 and 1:3, or 1:1 and 1:2. One skilled in the art may also contemplate chemical modifications to the DNA or RNA molecules. id="p-77" id="p-77" id="p-77" id="p-77" id="p-77"

[0077] In some embodiments a composi, tion provided herein that includes a mini- nucleosome core protein and/or a loaded mini-nucleosome) can be administered to or contacte d with a cell, tissue, or subject. The conditions of application may be in in vitro ex, vivo or in vivo. Such engineered cell may include a pharmaceutic alcarrier, e.g., that, may be used in, or is compatible with, deliver ofy therapeut materic ials (e.g., a. composition provided herein that includes a mini-nucleosom coree protein and/or a loaded, mini-nucleosome) to various parts of human body for example brain, retina, gut, pancreas, lung etc. without any limitations. id="p-78" id="p-78" id="p-78" id="p-78" id="p-78"

[0078] In some embodiments, a method of condensing a polynucleotide may include contacting a polynucleotide with a. mini-nucleosome core protein as described herein. The method may include process of neutralizi theng charge of a polynucleotide or condensation of the polynucleotide into nano-sized particles, including contacting the polynucleotide with a mini - nucleosome core protein described herein. id="p-79" id="p-79" id="p-79" id="p-79" id="p-79"

[0079] In some embodiments the, mini-nucleosom coree protein may be a branched peptide or a cyclic peptide but not. limited to these characteristics. One skilled in the art may contemplate changing the characteristics of mini-nucleosome core protein to obtain enhanced tropism to various cell types. id="p-80" id="p-80" id="p-80" id="p-80" id="p-80"

[0080] The present disclosure further provide as polynucleotide encoding an engineer ed, polypeptide (e.g., a. mini-nucleosom coree protein) as provided herein. The polynucleotide encoding the engineered polypeptide can be a DNA polynucleotide or an RNA polynucleotide.

In some instances, the present, disclosure provide as vector including a. polynucleotide that encodes an engineered polypeptide of the present disclosure. In some embodiments, the present disclosur providese a cell that includes a polynucleotide encoding an engineered polypeptid e (e.g., a. mini-nucleosome core protein) as provided herein, a. vecto includingr such polynucleot ide,or includes the sequenc ofe such polynucleotide. In certain embodiments, an engineered polypeptide of the present disclosur cane be isolated from one or more such cells. 21 id="p-81" id="p-81" id="p-81" id="p-81" id="p-81"

[0081] In various embodiments, one or more amino acids of an engineered polypeptid e of the present disclosure has at least 80% sequenc identite withy an amino acid sequenc selectede from SEQ ID NOs: 336-388 (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenc identie withty an amino acid sequenc selectede from SEQ ID NOs: 336-388). id="p-82" id="p-82" id="p-82" id="p-82" id="p-82"

[0082] In various embodiments, one or more amino acids of an engineered polypeptid e of the present disclosure (e.g., a mini-nucleosom coree protein) is pegylate acetd, ylated, methylated, glycosylated, phosphorylated, sumoylated, amidated, lipidated, prenylated, lipoyiate d,alkylat ed,acylated, glycated, nitrosylated, sulfated, carbamylated, carbonylated, neddylated biotinylate, ord, ribosylated.

DEFINITIONS id="p-83" id="p-83" id="p-83" id="p-83" id="p-83"

[0083] About: The term "about," when used herein in referenc toe a value, refers to a value that is similar, in context to the reference value.d In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" in that context. For example, in some embodiments the, term "about" may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.d id="p-84" id="p-84" id="p-84" id="p-84" id="p-84"

[0084] Administration: As used herein, the term "administration" typically refers to administration of a composition to a subject or system to achieve deliver ofy an agent that is, or is include in,d the composition. Those of ordinary skill in the art will be aware of a variet yof routes that may, in appropriate circumstances, be utilized for administrat ionto a subject for , example a human. For example, in some embodiments, administrat ionmay be ocular, oral, parenteral, topical etc., In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation) buccal,, dermal (which may be or include, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.) ,enteral, intra-arteri al, intradermal, intragastr ic,intramedulla intramusry, cular intra, nasal intrap, eriton eal,intratheca l, intravenous, intraventricular within, a specific organ (e. g. intrahepatic) mucosal,, nasal ,oral, rectal, subcutaneous, sublingual, topical tracheal, (e.g., by intratrache instilal lation) vaginal,, vitreal, etc. In some embodiments, administrat ionmay involve only a single dose. In some embodiments, administration may involv applicae tion of a fixed numbe ofr doses. In some 22 embodiments, administration may involv dosinge that is intermitt (e.g.,ent a plurality of doses separated in time and/or) periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. id="p-85" id="p-85" id="p-85" id="p-85" id="p-85"

[0085] Associated with: Two events or entiti arees "associated" with one anothe r,as that term is used herein, if the presence, level and/or form of one is correlated with that of the other.

For example, a particular entity (e.g., polypeptide, geneti signature,c metabolit microbe,e, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidenc ofe and/or susceptibili toty the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another .In some embodiments two, or more entiti thates are physically associated with one another are covalently linked to one anothe r;in some embodiments, two or more entiti thates are physically associated with one another are not covalently linked to one another but are non-covalently associated for, example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetis m,and combinations thereof. id="p-86" id="p-86" id="p-86" id="p-86" id="p-86"

[0086] Agent; As used herein, the term "agent," may refer to a compound, molecul ore, entity of any chemical class including, for example, a small molecul polypeptide,e, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term "agent" may refer to a compound, molecule, or entity that includes a polymer In. some embodiments, the term may refer to a compound or entity that includes one or more polymeric moieties In. some embodiments, the term "agent" may refer to a compound, molecule, or entity that is substantia llyfree of a particular polymer or polymeri moietc y. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantia llyfree of any polymer or polymeric moiety. id="p-87" id="p-87" id="p-87" id="p-87" id="p-87"

[0087] Amino acid: In its broadest sense, as used herein, "amino acid" refers to any compound and/or substanc thate can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurr ing 23 amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L- amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurrin gpeptides. "Nonstanda rdamino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natura source.l In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structur modificational as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylatio n,and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the genera structurel In. some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwis eidentical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwis eidentical unmodified amino acid. As will be clea rfrom context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide. id="p-88" id="p-88" id="p-88" id="p-88" id="p-88"

[0088] Between; As used herein, the term "between" refers to content that falls betwee n indicated upper and lower, or first and second, boundaries, inclusive of the boundaries. id="p-89" id="p-89" id="p-89" id="p-89" id="p-89"

[0089] Corresponding to; As used herein, the term "corresponding to" may be used to designa tethe position/identit of ay structur elemental in a compound or composition through comparison with an appropriate referenc compounde or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleot ide)may be identified as "corresponding to" a residue in an appropriate referenc polymere .For example, those of ordinary skill will appreciate that for , purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a referenc relatede polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190th amino acid in a particular amino acid chain but rather corresponds to the residu efoun dat 190 in the 24 referenc polypeptie de; those of ordinary skill in the art readily appreciate how to identif y "corresponding" amino acids. For example, those skilled in the art will be aware of various sequenc alignmente strategies, including softwar eprograms such as, for example, BLAST, CS- BLAST, CUDASW+, DIAMOND, FASTA, GGSEARCH/GL SEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Inferna KLASTl, , USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify "corresponding" residues in polypeptides and/or nucleic acids in accordance with the present disclosure. id="p-90" id="p-90" id="p-90" id="p-90" id="p-90"

[0090] Domain: The term "domain" as used herein refers to a section or portion of an entit y.In some embodiments, a "domain" is associated with a particular structur and/oral functional featur ofe the entity so that when, the domain is physically separated from the rest of its parent entity it, substantia llyor entirely retains the particular structur and/oral functional featur e.Alternatively or additionally, a domain may be or include a portion of an entity that, when separated from that (parent) entity and linked with a different (recipient) entit y, substantially retains and/or imparts on the recipient entity one or more structur and/oral functional features that characterized it in the parent entit y.In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nuclei acid,c or polypeptide) In. some embodiments a domain, is a section of a polypeptide; in some such embodiments, a domain is characterized by a particular structur elemental (e.g., a particul ar amino acid sequenc ore sequenc motife a, -helix character, B-sheet character ,coiled-coi l character, random coil character, etc.) ,and/or by a particular functional feature (e.g., binding activity, enzymat icactivity, folding activity, signaling activity, etc.). In some embodiments, a domain is or includes a characteristic portion or characteristic sequenc elemente . id="p-91" id="p-91" id="p-91" id="p-91" id="p-91"

[0091] Engineered; In general, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polynucleoti isde considered to be "engineered" when two or more sequences, that are not linked togethe inr that order in nature, are manipulated by the hand of man to be directl linkedy to one another in the engineered polynucleotide. Those of skill in the art will appreciate that an "engineered" nucleic acid or amino acid sequenc cane be a recombinant nucle icacid or amino acid sequence. In some embodiments, an engineered polynucleotide includes a domain-encoding sequenc regulatorye sequenc thate is found in nature in operative association with a first sequenc bute not in operative association with a second sequence, is linked by the hand of man so that it is operatively associated with the second sequence. Comparably ,a cell or organism is considered to be "engineered" if it has been manipulated so that its geneti informc ation is altered (e.g., new genetic material not previously present has been introduced, for example by transformati on, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deleti onmutation, or by mating protocols). As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as "engineered" even though the actual manipulation was performed on a prior entity. id="p-92" id="p-92" id="p-92" id="p-92" id="p-92"

[0092] Gene; As used herein, the term "gene" refers to a DNA sequenc thate codes for a product (e.g., an RNA product and/or a polypeptide product ).In some embodiments, a gene includes coding sequenc (i.e.,e sequenc thate encodes a particular product); in some embodiments, a gene includes non-coding sequence. In some particul arembodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that for, example, may control or impact one or more aspects of gene expression (e.g., a promoter) .A gene can be endogenous or non-endogen inous a particular context, e.g., a cell. A gene can be a transgene. id="p-93" id="p-93" id="p-93" id="p-93" id="p-93"

[0093] Gene product or expression product; As used herein, the term "gene product" or "expression product" generally refers to an RNA transcribed from a gene (pre-and/or post- processing) or a polypeptide (pre- and/or post-modificati on)encoded by an RNA transcribed from a gene. id="p-94" id="p-94" id="p-94" id="p-94" id="p-94"

[0094] Gm prove G " increase G " inhibit G or "reduce"; As used herein, the terms "improve," "increase" ,"inhibit," "reduce" ,or grammatical equivalents thereof, indicate values that are relative to a baseline or other referenc meae surement. In some embodiments, an appropriate referenc mease urement may be or include a measurement in a particular system (e.g., in a single individual) under otherwis ecomparabl econditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparabl e referenc agent.e In some embodiments, an appropriate referenc mease urement may be or include 26 a measurement in comparable system known or expecte tod respond in a particular way, in presence of the releva ntagent or treatment. id="p-95" id="p-95" id="p-95" id="p-95" id="p-95"

[0095] Increase and decrease ; As used herein, the terms "increase", "decrease," and grammatical equivalents thereof, indicate qualitative or quantitative difference from a reference. id="p-96" id="p-96" id="p-96" id="p-96" id="p-96"

[0096] Nucleic acid; As used herein, in its broadest sense, "nuclei acidc " refers to any compound and/or substanc thate is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substanc thate is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clea rfrom context, in some embodiments, "nucleic acid" refers to an individual nuclei acidc residu e(e.g., a nucleotide and/or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain including individual nuclei acidc residues. In some embodiments a "nucleic, acid" is or includes RNA; in some embodiments, a "nucleic acid" is or includes DNA. In some embodiments, a nucleic acid is, includes, or consists of one or more natura nucleicl acid residues. In some embodiments, a nucleic acid is, include ors, consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nuclei acidc in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, includes, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present disclosure.

Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, includes, or consists of one or more natura nucleosl ides (e.g., adenosine, thymidine, guanosin e,cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosin e,and deoxycytidine). In some embodiments, a nucleic acid is, includes, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine 3 -me, thyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5 -propynyl-cytidi ne,C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguani 2-ne, thiocytidin methylatede, bases, intercalate bases,d and combinations thereof ).In some embodiments, a nuclei acidc includes one or more modified sugars (e.g., 2'-fluororibose, ribose, 27 2'-deoxyribose, arabinose, and hexose )as compared with those in natura nucleicl acids. In some embodiments, a nucleic acid has a nucleotide sequenc thate encodes a functional gene product such as an RNA or protein. In some embodiments a nucleic, acid includes one or more introns.

In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerizati onbased on a complementary template (in vivo or in vitro), reproductio inn a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partl yor wholly single stranded; in some embodiments, a nucleic acid is partl yor wholly double strande d.

In some embodiments a nuclei acidc has a nucleotide sequenc includinge at least one eleme nt that encodes, or is the complement of a sequenc thate encode s,a polypeptide. In some embodiments, a nuclei acidc has enzymatic activity. id="p-97" id="p-97" id="p-97" id="p-97" id="p-97"

[0097] Operably linked; As used herein, "operably linked" refers to a juxtaposition where the component descris bed are in a relationship permitting them to function in thei rintended manne r.For example, a control element "operably linked" to a functional element is associated in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments "operabl, y linked" control elements are contiguous (e.g., covalently linked) with the coding elements of interest in; some embodiments, control elements act in trans to or otherwise at a from the functional eleme nt of interest. id="p-98" id="p-98" id="p-98" id="p-98" id="p-98"

[0098] Pharmaceutical composition; As used herein, the term "pharmaceutic al composition" refers to a composition in which an active agent is formulate togethed withr one or more pharmaceutically acceptable carriers. In some embodiments the, active agent is present in unit dose amount appropriate for administrat ionin a therapeutic regimen that shows a statisticall signifiy cant probability of achieving a predetermined therapeut effic ect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form ,including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or 28 suspensions), tablet e.g.,s, those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administrati on,for example, by subcutaneous, intramuscular intravenous, or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-rele patchase or spray applied to the skin, lungs or, oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublinguall y; ocularly; transdermal ly;or nasally, pulmonary, and to other mucosal surfaces. id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"

[0099] Polypeptide: As used herein, "polypeptide" refers to any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequenc thate occurs in nature. In some embodiments, a polypeptide has an amino acid sequenc thate does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may include or consist of natura aminol acids, non-natural amino acids, or both. In some embodiments, a polypeptide may include or consist of only natura aminol acids or only non-natural amino acids. In some embodiments, a polypeptide may include D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may include only D-amino acids. In some embodiments, a polypeptide may include only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, phosphorylation glycosylation,, glycation, sulfation, mannosylati on, nitrosylation, acylation, palmitoylation prenylat, ion, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may include a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not include any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptid e may be or include a staple polypeptidd e.In some embodiments, the term "polypeptide" may be appende tod a name of a referenc polypeptide,e activity, or structur ine; such instance its, is used herein to refer to polypeptides that share the relevant activity or structur ande thus can be considered to be members of the same class or family of polypeptides. For each such class ,the 29 present specificatio providesn and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are referenc polypeptidee fors the polypeptide class or family. In some embodiments, a member of a polypeptide class or famil yshows significant sequenc simile arity (e.g., homology) or identity with, shares a common sequenc motife (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class ;in some embodiments with all polypeptides within the class) . For example, in some embodiments, a member polypeptide shows an overall degree of sequenc similarie ty (e.g., homology) or identity with a referenc polypeptidee that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or include a characteristic sequence element) that shows very high sequenc identity,e often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments a useful, polypeptide may include or consist of a fragment of a parent polypeptid e.In some embodiments, a useful polypeptide as may include or consist of a plurality of fragments each, of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is foun din the polypeptide of interest (e.g., fragments that are directl linkedy in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent) so, that the polypeptide of interest is a derivative of its parent polypeptide. id="p-100" id="p-100" id="p-100" id="p-100" id="p-100"

[0100] Prevent or prevention ; As used herein, "prevent" or "prevention," when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristi csor symptoms of the disease, disorder or condition. Prevention may be considered complet whene onset of a disease, disorder or condition has been delayed for a predefined period of time. id="p-101" id="p-101" id="p-101" id="p-101" id="p-101"

[0101] Promoter; As used herein, a "promoter" or "promoter sequence" can be a DNA regulatory region that directly or indirectly (e.g., through promoter-bound proteins or substance s) participates in initiation and/or processivity of transcription of a coding sequence. A promoter may, under suitable conditions, initiate transcription of a coding sequenc upone bindin gof one or more transcription factors and/or regulator moietiesy with the promoter. A promoter that participates in initiation of transcription of a coding sequence can be "operably linke" dto the coding sequence. In certain instances, a promoter can be or include a DNA regulatory region that extend froms a transcription initiation site (at its 3’ terminus) to an upstream (5’ direction) position such that the sequenc soe designated includes one or both of a minimum number of bases or elements necessary to initiate a transcription event. A promoter may be, include, or be operably associated with or operabl ylinked to, expression control sequences such as enhancer and represso rsequences In. some embodiments, a promoter may be inducible. In some embodiments, a promoter may be a constitutive promoter. In some embodiments a conditional, (e.g., inducible) promoter may be unidirectional or bi-directional. A promoter may be or include a sequenc identicale to a sequenc knowne to occur in the genome of particular species. In some embodiments, a promoter can be or include a hybrid promoter, in which a sequenc containinge a transcriptional regulatory region can be obtained from one source and a sequenc containinge a transcription initiation region can be obtaine fromd a second source. Systems for linking control elements to coding sequenc withine a transgene are well known in the art (general molecular biologica andl recombinant DNA techniques are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). id="p-102" id="p-102" id="p-102" id="p-102" id="p-102"

[0102] Recombinant; As used herein, "recombinant" is intended to refer to polypeptide s that are designed, engineered, prepared, expressed, created, manufacture and/ord, or isolate byd recombinant means, such as polypeptides expressed using a recombinant expression vector transfecte intod a host cell; polypeptides isolate fromd a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit ,sheep, fish, etc) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene component thats encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s) or ,domain(s) thereof and/or; polypeptides prepared, 31 expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequenc elementse to one anothe r,chemicall synthesy izing selected sequence elements, and/or otherwis egenerating a nuclei acidc that encodes and/or direct expres ssion of the polypeptide or one or more component(s), portion(s), element(s) or ,domain(s) thereof. In some embodiments, one or more of such selected sequenc elementse is foun din nature. In some embodiments, one or more of such selected sequenc elementse is designed in silico. In some embodiments, one or more such selected sequence elements resul tsfrom mutagenesis (e.g., in vivo or in vitro) of a known sequenc element,e e.g., from a natura orl synthet sourceic such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc). id="p-103" id="p-103" id="p-103" id="p-103" id="p-103"

[0103] Reference: As used herein describes a standar dor control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequenc ore value of intere isst compared with a referenc ore control agent, animal indiv, idual, population, sample, sequenc ore value. In some embodiments, a referenc ore control is tested and/or determined substantia llysimultaneousl withy the testing or determination of interest In. some embodiments, a referenc ore control is a historical reference or contro l, optionall embodiey din a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficie nt similarities are present to justify reliance on and/or comparison to a particular possible reference or control. id="p-104" id="p-104" id="p-104" id="p-104" id="p-104"

[0104] Subject: As used herein, the term "subjec"t refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptibl toe a disease, disorder, or condition. In some embodiments, a subject displays one or more symptom sor characteristi csof a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characterist icof a disease , disorder, or condition. In some embodiments a subject, is someone with one or more features characteristic of susceptibili toty or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient In. some embodiments, a subject is an individual to whom diagnosi and/ors therapy is and/or has been administered. 32 id="p-105" id="p-105" id="p-105" id="p-105" id="p-105"

[0105] Substantial sequence similarity; The phrase "substantial sequence similarity" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciate byd those of ordinary skill in the art, two sequences are generally considered to be "substantia llysimilar" if they contain a conservative amino acid substitution in corresponding positions. A conservativ substite ution is one in which an amino acid has been replaced by a non- identical residu ehaving appropriately similar structur and/oral functional characteristics. For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "non- polar" side chains. Substitut ionof one amino acid for another of the same type may often be considered a conservativ substitution.e Typical amino acid categorizations are summarized in Tables 1 and 2 below: Table 1 Alanine Ala A nonpolar neutral 1.8 Arginine Arg R polar positive -4.5 Asparagine Asn N polar neutral -3.5 Aspartic acid Asp D polar negative -3.5 Cysteine Cys C nonpolar neutral 2.5 Glutamic acid Glu E polar negative -3.5 Glutamine Gin polar neutral -3.5 Q nonpolar Glycine Gly G neutral -0.4 Histidine His H polar positive -3.2 Isoleucine lie I nonpolar neutral 4.5 L nonpolar 3.8 Leucine Leu neutral Lysine Lys K polar positive -3.9 Methionine Met M nonpolar neutral 1.9 PhenylalaninePhe F nonpolar neutral 2.8 Proline Pro P nonpolar neutral -1.6 Serine Ser S polar neutral -0.8 33 Thr T polar Threonine neutral -0.7 Tryptophan Trp W nonpolar neutral -0.9 Tyrosine Tyr Y polar neutral -1.3 Valine Vai V nonpolar neutral 4.2 Table 2 Ambiguous Amino Acids 3-Letter !-Letter Asparagine or aspartic acid Asx B Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xie J Unspecified or unknown amino acid Xaa X id="p-106" id="p-106" id="p-106" id="p-106" id="p-106"

[0106] As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variet yof algorithms, including those available in commercial compute r programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI- BLAST for amino acid sequence s.Exemplary such programs are described in Altschul et, al., Basic local alignmen seart ch tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul et, al., Methods in Enzymology; Altschu etl, al., "Gapped BLAST and PSI-BLAST: a new generation of protein databas esearch programs," Nuclei Acidsc Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformati cs: A Practical Guide to the Analysi sof Genes and Proteins, Wiley, 1998; and Misener et, al., (eds.), Bioinformati csMethods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying similar sequences, the programs mentioned above typically provide an indication of the degree of similarity In. some embodiments, two sequences are considered to be substantia llysimilar if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of thei rcorresponding residues are similar and/or identical over a relevant stretch of residues. In some embodiments, the releva ntstretch is a complet sequence.e In some embodiments, the relevant stretch is at least 10, at least 15, at least , at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at 34 least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more residues. As would be appreciate byd one of ordinary skill in the art sequences with substanti sequencal similarie ty may be homologs of one another. id="p-107" id="p-107" id="p-107" id="p-107" id="p-107"

[0107] Substantial sequence identity: As used herein, the phrase "substantial sequence identit" refy ers to a comparison between amino acid or nucle icacid sequence s.As will be appreciate byd those of ordinary skill in the art, two sequences are generally considered to be "substantia llyidentical" if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences .

Exemplary such programs are described in Altschul et al., Basic local alignmen seart ch tool, J.

Mol. Biol. ,215(3): 403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysi sof Genes and Proteins, Wiley, 1998; and Misener et, al, (eds.) ,Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the programs mentioned above typicall providey an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of thei rcorresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complet e sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues. id="p-108" id="p-108" id="p-108" id="p-108" id="p-108"

[0108] Therapeutic agent: As used herein, the phrase "therapeutic agent" in general refers to any agent that elicit as desired pharmacologic aleffect when administered to an organism .In some embodiments, an agent is considered to be a therapeut agentic if it demonstrates a statistica llysignificant effe ctacross an appropriate populatio n.In some embodiments, the appropriate populatio mayn be a populatio ofn model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender genetic, background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorat e,relieve, inhibit, prevent, delay onset of, reduce severit of,y and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments a ",therapeut agentic " is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent for which a medica lprescription is required for administrat ionto humans. id="p-109" id="p-109" id="p-109" id="p-109" id="p-109"

[0109] Therapeutic regimen; A "therapeutic regimen," as that term is used herein, refer s to a dosing regime nwhose administration across a relevant population may be correlated with a desire dor benefici altherapeut outcome.ic id="p-110" id="p-110" id="p-110" id="p-110" id="p-110"

[0110] Therapeutically effective amount: As used herein, is meant an amount that produce thes desired effe ctfor which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder and/or, condition in accordance with a therapeutic dosing regimen, to trea tthe disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successfu treatl ment be achieve din a particular individu al.Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacologic alresponse in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, referenc toe a therapeutically effective amount may be a referenc toe an amount as measured in one or more specific tissue (e.g.,s a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva ,serum, sweat ,tears, urine, etc.). Those of ordinary skill in the art will appreciate that in, some embodiments a thera, peutica llyeffective amount of a particular agent or therapy may be formulate and/ord administered in a single dose. In some embodiments a , therapeutically effective agent may be formulate and/ord administered in a plurality of doses, for example, as part of a dosing regimen. 36 id="p-111" id="p-111" id="p-111" id="p-111" id="p-111"

[0111] Treatment; As used herein, the term "treatment" (also "trea"t or "treating") refers to any administrat ionof a therapy that partially or completel alleviay tes, ameliorates, relives, inhibits, delays onset of, reduces severit of,y and/or reduces incidence of one or more symptoms , feature and/ors, causes of a particula diseasr e, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subjec twho exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subjec twho has been diagnosed as suffering from the relevant disease , disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibili factorsty that are statistica llycorrelated with increased risk of development of the relevant disease, disorder, and/or condition. id="p-112" id="p-112" id="p-112" id="p-112" id="p-112"

[0112] Variant: As used herein in the context of molecule e.g.,s, nuclei acids,c proteins, or small molecules, the term "variant" refers to a molecule that shows significant structur al identity with a referenc moleculee but differs structurall fromy the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the referenc entie ty. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particula moleculr ise properly considered to be a "variant" of a referenc moleculee is based on its degree of structur identityal with the referenc molecule.e As will be appreciated by those skilled in the art, any biologica orl chemical referenc moleculee has certain characteristic structur elements.al A variant, by definition, is a distinct molecule that shares one or more such characterist icstructural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequenc elemente include ofd a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biologica functl ion; a nuclei acidc may have a characteristic sequenc elemee nt included of a pluralit ofy nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nuclei acidc may differ from a referenc polypeptidee or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., 37 carbohydrates, lipids, phosphate groups) that are covalently component ofs the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequenc identitye with a referenc polypeptidee or nuclei acidc that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequenc elemente with a reference polypeptide or nucleic acid. In some embodiments, a referenc polypeptide ore nuclei acidc has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the referenc polypeptidee or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activiti esof the referenc polypeptidee or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activiti esas compared to the referenc polypeptidee or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of a referenc polypeptide ore nucleic acid if it has an amino acid or nucleotide sequenc thate is identical to that of the referenc bute for a smal lnumbe ofr sequenc alterae tions at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substitute inserted,d, or delete asd, compared to the reference. In some embodiments, a variant polypeptid ore nucleic acid includes about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often a, variant polypeptide or nucleic acid includes a very smal lnumbe (e.g.,r fewer than about 5, about 4, about 3, about 2, or about 1) numbe ofr substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particula r biologica activil ty) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid includes not more than about 5, about 4, about 3, about 2, or about 1 addition or deleti on,and, in some embodiments includes, no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid includes fewer than about , about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference 38 polypeptide or nucleic acid is one found in nature. In some embodiments, a reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

BRIEF DESCRIPTION OF THE DRAWING id="p-113" id="p-113" id="p-113" id="p-113" id="p-113"

[0113] Figure 1 includes panel A and panel B. Panel A is a schematic representat ionof how a mini-nucleosom coree protein modified with PEG12, shown in Panel B, at a lysine residu e can undergo a condensation reaction with a DNA molecule to produce a loaded mini- nucleosome. Each nucleic acid molecule may require several (1 to 1000) mini-nucleosome core proteins to neutral izethe negative charges in the DNA to form a loaded mini-nucleosome. The schematic is intended only as a cartoon diagram, and is not intende tod be representat iveof the actua lstructure of loaded mini-nucleosomes except to the extent that loaded mini-nucleosome includes nucleic acids associated with core proteins. id="p-114" id="p-114" id="p-114" id="p-114" id="p-114"

[0114] Figure 2 is a chart showing data obtained from mass spectromet analysesry after the formulation of the mini-nucleosom coree protein modified with PEG12 at the firs tlysine residu ein the sequence. id="p-115" id="p-115" id="p-115" id="p-115" id="p-115"

[0115] Figure 3 is a schematic representat ionof how a mini-nucleosom coree protein modified with IkDa PEG at a lysine residue can undergo a condensation reaction with a DNA molecule to produce a loaded mini-nucleosome Figure. 3 includes panel A and panel B. Panel A is a schemati crepresentati ofon how a mini-nucleosome core protein modified with IkDa PEG, shown in Pane lB, at a lysine residue can undergo a condensation reaction with a DNA molecul e to produce a loaded mini-nucleosome Each. nuclei acidc molecule may require several (1 to 1000) mini-nucleosom coree proteins to neutral izethe negativ chargese in the DNA to form a loaded mini-nucleosome. The schematic is intende onlyd as a cartoon diagram, and is not intended to be representati ofve the actual structure of loaded mini-nucleosomes except to the extent that loaded mini-nucleosom includese nucleic acids associated with core proteins. id="p-116" id="p-116" id="p-116" id="p-116" id="p-116"

[0116] Figure 4 includes panel A and panel B. Panel A is a schematic representat ionof how a mini-nucleosom coree protein modified with 2kDa PEG, shown in Pane lB, at a lysine residu ecan undergo a condensation reaction with a DNA molecule to produce a loaded mini- nucleosome. Each nucleic acid molecule may require several (1 to 1000) mini-nucleosome core proteins to neutral izethe negative charges in the DNA to form a loaded mini-nucleosome. The 39 schematic is intended only as a cartoon diagram, and is not intende tod be representat iveof the actua lstructure of loaded mini-nucleosomes except to the extent that loaded mini-nucleosome includes nucleic acids associated with core proteins. id="p-117" id="p-117" id="p-117" id="p-117" id="p-117"

[0117] Figure 5 includes panel A and panel B. Panel A is a schematic representat ionof how a mini-nucleosom coree protein modified with 5kDa PEG, shown in Pane lB, at a lysine residu ecan undergo a condensation reaction with a DNA molecule to produce a loaded mini- nucleosome. Each nucleic acid molecule may require several (1 to 1000) mini-nucleosome core proteins to neutral izethe negative charges in the DNA to form a loaded mini-nucleosome. The schematic is intended only as a cartoon diagram, and is not intende tod be representat iveof the actua lstructure of loaded mini-nucleosomes except to the extent that loaded mini-nucleosome includes nucleic acids associated with core proteins. id="p-118" id="p-118" id="p-118" id="p-118" id="p-118"

[0118] Figure 6 includes panel A and panel B. Panel A is a schematic representat ionof how a mini-nucleosom coree protein modified with lOkDa PEG, shown in panel B, at a lysine residu ecan undergo a condensation reaction with a DNA molecule to produce a loaded mini- nucleosome. Each nucleic acid molecule may require several (1 to 1000) mini-nucleosome core proteins to neutral izethe negative charges in the DNA to form a loaded mini-nucleosome. The schematic is intended only as a cartoon diagram, and is not intende tod be representat iveof the actua lstructure of loaded mini-nucleosomes except to the extent that loaded mini-nucleosome includes nucleic acids associated with core proteins. id="p-119" id="p-119" id="p-119" id="p-119" id="p-119"

[0119] Figure 7 is a set of images that includes panels A, B, and C, each of which presents an image from Transmission Electron Microscopy (TEM) of loaded mini-nucleosomes. id="p-120" id="p-120" id="p-120" id="p-120" id="p-120"

[0120] Figure 8 is a graph showing concentration of expressed Factor 8 protein as measured by Elisa. id="p-121" id="p-121" id="p-121" id="p-121" id="p-121"

[0121] Figure 9 is a set of images including panels A, B, and C, each of which is a fluorescent microscopy image that illustra tesgene expression in liver tissue of proteins encoded by nucleic acids present in loaded mini-nucleosomes. id="p-122" id="p-122" id="p-122" id="p-122" id="p-122"

[0122] Figure 10 is a set of images including panels A, B, C & D each of which is a fluorescent microscopy image that illustra tesgene expression in mice RPE tissue of proteins encoded by nucleic acids present in loaded mini-nucleosomes. Panel A is a retinal section that demonstrates RPE specific expression. Panels B is a RPE wholemount that demonstrate RPEs 40 specific expression. Panels B and D represent untreated contro lsamples of a retina section and RPE wholemount respectively. id="p-123" id="p-123" id="p-123" id="p-123" id="p-123"

[0123] Figure 11 is a set of images including panels A, B, C & D each of which is a fluorescent microscopy image that illustra tesgene expression in rat retinal tissue of proteins encoded by nucleic acids present in loaded mini-nucleosomes. Panels A and C are retina l sections that demonstrate RPEs specific expression and panels B and D present plasmid injected control samples. id="p-124" id="p-124" id="p-124" id="p-124" id="p-124"

[0124] Figure 12 is a set of images including panels A, B, C & D each of which is a fluorescent microscopy image that illustra tesgene expression in mice retina tissuel of proteins encoded by nucleic acids present in loaded mini-nucleosomes. Panel A is a retinal section that demonstrates GFP expression in retinal neurons. Panel C is a retinal wholemount that demonstrates GFP expression in retinal photoreceptors. Panels B and D represent untreated control sample sof a retinal section and RPE wholemount respectively. id="p-125" id="p-125" id="p-125" id="p-125" id="p-125"

[0125] Figure 13 is a set of images including panels A, B & C each of which is a fluorescent microscopy image that illustra tesgene expression in mice lung of proteins encoded by nucleic acids present in loaded mini-nucleosomes. Panel A demonstrates GFP expression in alveoli and bronchioles. Panel B demonstrate CFTRs staining. Panel C is a merge for panels A and B demonstrating colocalization of GFP and CFTR staining. id="p-126" id="p-126" id="p-126" id="p-126" id="p-126"

[0126] Figure 14 is a set of images including panels A, B & C each of which is a fluorescent microscopy image at higher magnificati thaton illustrates gene expression in mice lung epithelium of proteins encoded by nucleic acids present in loaded mini-nucleosomes. Panel A demonstrate GFPs expression in alveo liand bronchioles. Panel B demonstrates CFTR staining. Pane lC is a merge for panels A and B demonstrating colocalization of GFP and CFTR including DAPI staining. id="p-127" id="p-127" id="p-127" id="p-127" id="p-127"

[0127] Figure 15 is a set of image sthat illustrates gene expression in mice whole lung tissue of proteins encoded by nucleic acids present in loaded mini-nucleosomes. id="p-128" id="p-128" id="p-128" id="p-128" id="p-128"

[0128] Figure 16 is a set of image sincluding panels A, B & C that illustrates gene expression in mice brain, gut and pancreas tissue of proteins encoded by nuclei acidsc present in loaded mini-nucleosomes. Panel A demonstrate expres ssion patter inn olfactory neurons. Panel B 41 and its inset below demonstrate expres ssion pattern in small intestine. Panel C and its inset below demonstrates expression patter inn pancreas. id="p-129" id="p-129" id="p-129" id="p-129" id="p-129"

[0129] Figure 17 is a set of image sincluding panels A, B & C that illustrates gene expression in mice tracheal tissue of proteins encoded by nucleic acids present in loaded mini- nucleosomes. Panel A demonstrates GFP expression in tracheal epithelium and inner tracheal muscle. Panel B demonstrates dystrophin staining patte rnin expression in inner and outer tracheal muscle. Pane lC is a merge of panel A and B that demonstrates colocalization of dystrophin staining patter withn GFP in inner tracheal muscle cells. id="p-130" id="p-130" id="p-130" id="p-130" id="p-130"

[0130] Figure 18 is a set of image sincluding panels A, B & C that illustrates gene expression in mice muscle tissue of proteins encoded by nucle icacids present in loaded mini - nucleosomes. Panel A demonstrates GFP expression in mouse muscle cells. Panel B demonstrates dystrophin staining patter inn expression in mouse muscle cells. Panel C is a merge of panel A and B that demonstrate colocalizats ion of dystrophin staining patter withn GFP in mouse muscle cells. id="p-131" id="p-131" id="p-131" id="p-131" id="p-131"

[0131] Figure 19 is a graph showing increase in concentration of expressed Factor 8 protein as measured by Elisa following a first dose and a second dose suggesting lack of neutralizing effect or in other words lack neutralizing antibody activity. id="p-132" id="p-132" id="p-132" id="p-132" id="p-132"

[0132] Figure 20 includes panels A, B, C, and D. Panel A is a schematic representation of an unmodified mini-nucleosome core protein. Panel B is a schemati crepresentati ofon a modified mini-nucleosom coree protein, where an asparagine residu e(N) is modified with an unbranche modificationd chain including GlcNac, GlcNac ,and sialic acid. Panel C is a schematic representation of a modified mini-nucleosom coree protein, where an asparagine residu e(N) is modified with a branched modification chain including a trunk including GlcNac , a branch including GlcNac and sialic acid, and a branch including fucose .Panel D is a schematic representation of a modified mini-nucleosom coree protein, where an asparagine residu e(N) is modified with a branched modification chain including a trunk including GlcNac , a branch including fucose ,and a branch including a secondary trunk including GlcNac, a secondary branch including sialic acid, and a secondary branch including mannose. id="p-133" id="p-133" id="p-133" id="p-133" id="p-133"

[0133] Figure 21 includes panels A, B, C, D, E, F and G. Panel A is a schematic representati ofon an unbranche modificationd chain (C14010N1H23) including GlcNac and 42 mannose that could, e.g., modif yan asparagine residu ein a mini-nucleosom coree protein. Panel B is a schematic representat ionof an unbranched modification chain (C25018N2H40) including GlcNac, mannose, and fucose that could, e.g., modify an asparagine residue in a mini- nucleosome core protein. Panel C is a schemati crepresentati ofon a di-antennal branched modification chain (C34025N2H56) including a trunk including GlcNac, GlcNac ,and Mannose and two branches, each including mannose, which modification chain could modify, e.g., an asparagine residue in a mini-nucleosom coree protein. A modification chain having this structure can be referred to as low mannose. Panel D is a schematic representat ionof di-antennal branched modification chain (C64049N4H106) that includes a trunk including GlcNac ,a branch including fucose, and a branch including a secondary trunk including GlcNac and mannose and two secondary branches each including three mannose modifications, which modification chain could, e.g., modify an asparagine residu ein a mini-nucleosom coree protein. A modification chain having this structur cane be referred to as high mannose. Panel E is a schematic representati ofon a di-antennary silalylated & fucosylate branched d modification chain (C90065N6H146) that includes a trunk including GlcNac ,a branch including fucose ,and a branch including a secondary trunk including GlcNac and mannose, two secondary branches each including mannose, GlcNac ,mannose, and fucose, which modification chain could, e.g., modif y an asparagine residu ein a mini-nucleosome core protein. Panel Fisa schematic representation of tri-antennar silalylay ted and fucosylated branched modification chain (C115083N8H186) that that includes a trunk including GlcNac, a branch including fucose, and a branch including a secondary trunk including GlcNac and mannose with two secondary branches. One of the secondary branches includes mannose, GlcNac, mannose, and fucose, while the other includes a tertiary trunk including mannose and, two tertiary branches each including GlcNac ,mannose, and fucose. The branched modification chain could, e.g., modif yan asparagine residu ein a mini-nucleosom coree protein. Panel G is a schematic representation of tetra-antennary sialylat ed& fucosylate branchedd modification chain (C1400101N10H226) that includes a trunk including GlcNac, a branch including fucose ,and a branch including a secondary trunk including GlcNac and mannose with two secondary branches. Each of the secondary branches includes a tertiary trunk including mannose and, two tertiary branches each including GlcNac ,mannose, 43 and fucose. The branched modification chain could, e.g., modif yan asparagine residu ein a mini-nucleosome core protein. id="p-134" id="p-134" id="p-134" id="p-134" id="p-134"

[0134] Figure 22 includes panels A, B, C, D, E, F, G, H, I, and J. Panel A is a schematic representati ofon a GlcNac modification that could modif ya serin eresidue of a mini-nucleoso me core protein. Panel B is a schematic representati ofon an unbranche glycand modification chain including GalNac and galactose. Panel C is a schematic representat ionof a di-antennary branched glycan modification including a GalNac trun k,a GlcNac branch, and a galactose branch, which branched modification could modify a serin eresidue of a mini-nucleosome core protein. Pane lD is a schematic representati ofon an unbranched modification including mannose, GlcNac, galactose, and NeuAc ,which unbranched modification could modif ya serine residu eof a mini-nucleosome core protein. Panel E is a schematic representation of an unbranche modificationd including fucose, GlcNac ,galactose, and sialic acid, which unbranche d modification could modif ya serine residu eof a mini-nucleosome core protein. Panel Fisa schematic representat ionof a di-antennar branchedy modification including a GalNac trunk, a galactose branch, and a branch including GlcNac, galactose, GlcNac ,galactose, and NeuAc, which branched modification could modify a serine residu eof a mini-nucleosome core protein.

Panel G is a schemati crepresentati ofon a di-antennar branchey d modification including a GalNac trunk, a galactos branch,e and branch including a secondary trunk including GlcNac , galactose, GlcNac, and galactose with a fucos esecondary branch and a NeuAc secondary branch, which branched modification could modify a serin eresidue of a mini-nucleosome core protein. Panel H is a schematic representati ofon single sugar addition that could modif ya serine or threonine, which single sugar modification could be, e.g., a galactose, glucose mannose,, fucose ,or sialic acid. Pane llisa schematic representati ofon an unmodified mini-nucleoso me core protein. Panel J is a schematic representati ofon a modified mini-nucleosome core protein, where a serine residue is modified with an unbranched glycan modification including GalNac , GalNac, and galactose. id="p-135" id="p-135" id="p-135" id="p-135" id="p-135"

[0135] Figure 23 includes panels A, B, C, D, and E. Panel A is a schematic representati ofon an unmodified mini-nucleosome core protein. Panel B is a schematic representati ofon a modified mini-nucleosome core protein, where a lysine residu e(K) is modified with an acetyl group. Panel C is a schematic representation of a modified mini 44 nucleosome core protein, where two lysine residuess (K) are each modified with an acetyl group.

Panel D is a schematic representati ofon a modified mini-nucleosome core protein, where each of a lysine residue (K) and a valine (V) is modified with an acetyl group. Panel E is a schematic representati ofon a modified mini-nucleosome core protein, where each of a lysine residue (K) and an alanine (A) residue is modified with an acetyl group. id="p-136" id="p-136" id="p-136" id="p-136" id="p-136"

[0136] Figure 24 is a schematic representati ofon an acetylated lysine. id="p-137" id="p-137" id="p-137" id="p-137" id="p-137"

[0137] Figure 25 includes panels A, B, and C. Panel A is a schematic representati ofon an unmodified mini-nucleosome core protein. Panel B is a schematic representat ionof a modified mini-nucleosome core protein, where a tyrosine residu e(Y) is modified with a sulfa te group. Panel C is a schemati crepresentati ofon a sulfate tyrosined id="p-138" id="p-138" id="p-138" id="p-138" id="p-138"

[0138] Figure 26 includes panels A, B, C, and D. Panel A is a schematic representation of an unmodified mini-nucleosome core protein. Panel B is a schemati crepresentati ofon a modified mini-nucleosom coree protein, where a cysteine residu e(C) is modified with prenyl group. Panel C is a schemati crepresentati ofon a farnesyl group linked with a polypeptide.

Panel D is a schemati crepresentati ofon a geranylgeranyl group linked with a polypeptide. id="p-139" id="p-139" id="p-139" id="p-139" id="p-139"

[0139] Figure 27 includes panels A, B, C, D, E, and F. Pane lA is a schematic representati ofon an unmodified mini-nucleosome core protein. Panel B is a schematic representati ofon a modified mini-nucleosome core protein, where a serin eresidue (S) is modified with a phospho group. Pane lC is a schematic representat ionof phosphotyrosine.

Panel D is a schemati crepresentati ofon phosphoserine. Panel E is a schemati crepresentati ofon phosphothreonine. Panel F is a schematic representat ionof bis-phosphohistidine. id="p-140" id="p-140" id="p-140" id="p-140" id="p-140"

[0140] Figure 28 includes panels A, B, C, D, and E. Panel A is a schematic representati ofon an unmodified mini-nucleosome core protein. Panel B is a schematic representati ofon a modified mini-nucleosome core protein sequence, where a lysine residu e(K) is methylated. Pane lC is a schematic representat ionof mono-methyl lysine. Panel D is a schematic representation of di-methyl lysine. Panel E is a schemati crepresentati ofon tri-methyl lysine. id="p-141" id="p-141" id="p-141" id="p-141" id="p-141"

[0141] Figure 29 includes panels A, B, C, and D. Panel A is a schematic representation of an unmodified mini-nucleosome core protein. Panel B is a schemati crepresentati ofon a modified mini-nucleosome core protein, where a proline residue (P) is hydroxylated Panel. C is 45 a schematic representat ionof 4- hydroxylated proline. Pane lD is a schemati crepresentati ofon 3-hydroxylated proline. id="p-142" id="p-142" id="p-142" id="p-142" id="p-142"

[0142] Figure 30 includes panels A, B, C, D and E. Panel A is a schematic representati ofon an unmodified mini-nucleosome core protein. Panel B is a schematic representati ofon a modified mini-nucleosome core protein, where a lysine residu e(K) is lipidated and/or lipoylate d.Pane lC is a schematic representat ionof S-myristoylated glycine.

Panel D is a schematic representati ofon S-palmitoylated cysteine. Pane lE is a schematic representati ofon O-palmitoylated cysteine. id="p-143" id="p-143" id="p-143" id="p-143" id="p-143"

[0143] Figure 31 includes panels A and B. Panel A is an IVIS® Spectrum In Vivo Imaging System (IVIS) image of a mouse administered an unmodified mini-nucleosom coree protein according to SEQ ID NO: 399 loaded with a nuclei acidc payload including a gene encoding luciferase as a representati expreve ssion product Panel. B is an IVIS® Spectrum In Vivo Imaging System (IVIS) image of a mouse administered a phosphorylated mini-nucleoso me according to SEQ ID NO: 399 loaded with a nucleic acid payload including a gene encoding luciferase as a representati expressive on product Panel. B shows that the modified mini - nucleosome core protein, but not the unmodified mini-nucleosome core protein, resul tsin robust expression of the representati nucleicve acid payload-encoded expression product (here, luciferase) in certain tissues including central nervou systems cells including neurons, and including spinal cord cells and brain neurons. id="p-144" id="p-144" id="p-144" id="p-144" id="p-144"

[0144] Figure 32 includes panels A and B. Panel A is an IVIS® Spectrum In Vivo Imaging System (IVIS) image of a mouse administered an unmodified mini-nucleosom coree protein according to SEQ ID NO: 388 loaded with a nuclei acidc payload including a gene encoding luciferase as a representati expreve ssion product Panel. B is an IVIS® Spectrum In Vivo Imaging System (IVIS) image of a mouse administered a sulfated mini-nucleoso me according to SEQ ID NO: 388 loaded with a nucleic acid payload including a gene encoding luciferase as a representati expressive on product Panel. B shows that the modified mini - nucleosome core protein, but not the unmodified mini-nucleosome core protein, resul tsin robust expression of the representati nucleicve acid payload-encoded expression product (here, luciferase) in certain tissues including central nervou systems cells including neurons, and including spinal cord cells and brain neurons. 46 id="p-145" id="p-145" id="p-145" id="p-145" id="p-145"

[0145] Figure 33 includes panels A, B, C, D and E. Panel A is an IVIS® Spectrum In Vivo Imaging System (IVIS) image of a mouse administered a sulfated mini-nucleoso me according to SEQ ID NO: 388 loaded with a nucleic acid payload including a gene encoding luciferase as a representati expressive on product Panel. A shows a high degree of expression in certain tissues including central nervous system cells including neurons, and including spinal cord cells and brain neurons. Panels B, C, D, and E are image sof a representati tissueve section from the brain of an animal shown in Figure A (left) each, of which represents a differe nt imaging or overlay. Panel B shows a luciferase stain. Panel C shows an anti-NeuN (neuronal marker )antibody stain. Pane lD shows a D-D API (nuclear) stain. Panel E shows an overla yof the luciferase, anti-NeuN antibody and, D-D API stains. Images demonstrate robust expression of the representati nucleicve acid payload-encoded expression product (here, luciferas e)in brain cells, particularly including brain neuron s,when the mini-nucleosome is sulfate butd not when the mini-nucleosome is unmodified. id="p-146" id="p-146" id="p-146" id="p-146" id="p-146"

[0146] Figure 34 includes panels A, B, C, D, E, and F, each of which shows a retinal wholemount. Panels A, B, and C show a representati retive nal wholemount from a mouse administered an unmodified mini-nucleosome core protein according to SEQ ID NO: 401 loaded with a nucleic acid payload including a gene encoding green fluorescent protein (GFP) as a representati expreve ssion product .Panel A shows native GFP fluorescence. Panel B shows an anti-GFP antibody stain. Pane lC shows an overla yof native GFP fluorescen andce anti-GF P antibody stain. Panels D, E, and F show a representat iveretinal wholemount from a mouse administered an acetylated mini-nucleosome according to SEQ ID NO: 401 loaded with a nucleic acid payload including a gene encoding GFP as a representati expressive on product.

Panel D shows native GFP fluorescence. Panel E shows an anti-GFP antibody stain. Panel F shows an overla yof native GFP fluorescen andce anti-GFP antibody stain. Images demonstrate robust expression of the representati nucleicve acid payload-encoded expression product (here, GFP) in retinal cells, in particular photoreceptor whens, the mini-nucleosome is acetylated but not when the mini-nucleosome is unmodified. id="p-147" id="p-147" id="p-147" id="p-147" id="p-147"

[0147] Figure 35 includes panels A, B, C, and D, each of which is an image of a representati retive nal wholemount from a mouse administered a mannosylated mini-nucleosome according to SEQ ID NO: 447 loaded with a nucleic acid payload including a gene encoding 47 GFP as a representati expreve ssion product. Panel A shows native GFP fluorescence. Pane lB shows an anti-GFP antibody stain. Panel C shows a peanut agglutinin (PNA; marker for photoreceptors) stain. Panel D shows an overla yof native GFP fluorescence, anti-GF Pantibody stain, and PNA stain. Images demonstrate robust expression of the representati nucleicve acid payload-encoded expression product (here, GFP) in retinal cells, in particular photoreceptors, when the mini-nucleosome is mannosylated. An unmodified control loaded with the same nucleic acid payload was not robustly expressed in retinal cells or photoreceptors.

DETAILED DESCRIPTION id="p-148" id="p-148" id="p-148" id="p-148" id="p-148"

[0148] The present disclosure provides, among other things, methods and compositions relating to mini-nucleosome core proteins and uses thereof Mini-. nucleoso mecore proteins disclosed herein include, among other things, (a) a nucleic acid bindin gdomain (NABD), (b) a targeting domain and/or (c) a nucleic acid release domain, and/or a stability domain, and/or an oligomerization domain, and/or a linker domain. Certain mini-nucleosom coree proteins disclosed herein include (a) a nucleic acid bindin gdomain (NABD); (b) a targeting domain; (c) a nucleic acid release domain; and, optionally, (d) further domains including, e.g., one or more of a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, mini-nucleosome core protein of the present disclosure is a modified mini-nucleosome core protein, i.e., a mini-nucleosome core protein that is modified in that it includes one or more modified amino acid residue s,such as an amino acid residu emodified to include a modification provided herein, including without limitation any of one or more of (i) phosphorylation; (ii) sulfation; (iii) glycosylation (e.g., N-glycosylati on,C-glycosylation, and/or O-glycosylation); (iv) prenylation (e;g., geranylat ionand/or famesylation) (v); methylation; (vi); sialylation; (vii) lipidation and/or lipoylation;; (viii) acetylation; (ix) hydroxylation;; (x) palmitoylation; (xi) mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylation; and/or; (xv) any combination thereof, including any numbe ofr one or more of the modifications or variants thereof, e.g., in a branched or unbranche modificationd chain. In various embodiments, a mini- nucleosome core protein of the present disclosure can be a modified mini-nucleosom coree protein that includes (a) a nucleic acid bindin gdomain ("NABD"); (b) a targeting domain; (c) a modified amino acid residue and,; optionally, (d) further domains including, e.g., one or more of 48 a nucleic acid release domain, a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, a mini-nucleosom coree protein of the present disclosure can be a modified mini-nucleosome core protein that includes (a) a nucleic acid bindin gdomain ("NABD"); (b) a targeting domain; (c) a nucleic acid release domain; (d) a modified amino acid residue and,; optionally, (e) further domains including, e.g., one or more of a stability domain, an oligomerization domain, and/or a linker domain. In various embodiments, a mini-nucleoso me core protein is associated with one or more nucle icacid molecules, thereby forming a loaded mini-nucleosom coree protein (the mini-nucleosom coree protein and associated nucle icacid molecules also referred to together herein as a loaded mini-nucleosome) In. various embodiments, a loaded mini-nucleosom includese two or more mini-nucleosome core proteins and one or more nucleic acid molecules. In various embodiments, a loaded mini-nucleosome is administered to a subjec tin need thereof. id="p-149" id="p-149" id="p-149" id="p-149" id="p-149"

[0149] Polynucleotide chains typicall carry y phosphates with negativ charge.e Accordingl y,positive charges in proteins such as histones help condense nucleic acids. The present disclosure appreciates that nucleic acid bindin gdomains, derived, e.g., from histones can, be utilized in artificial lyconstructed mini-nucleosome core proteins as a non-viral proteinaceous vector. id="p-150" id="p-150" id="p-150" id="p-150" id="p-150"

[0150] Most mammalian cells possess cell surface bindin gmoieties or receptors that recognize (and/or are recognize by),d bind, and internalize molecules or entities like viruses and bacteria. Various compositions and methods disclosed herein make use of such cell surfac e bindin gmotif ins combination with nucleic acid binding domains and poly-Arginine domains in a mini-nucleosome core protein. In various embodiments, a mini-nucleosome core protein is capable of condensing or, participating in or facilitatin theg condensation of, one or more nucleic acids. In various embodiments, a mini-nucleosom coree protein facilitates internalization of associated nucleic acids, e.g., in a loaded mini-nucleosome, into specific cell types, e.g., via endocytosis or via other cellula entryr mechanisms. Accordingly, in various embodiments, the present disclosure includes mini-nucleosome core proteins that incorporate targeting moieties capable of bindin withg cell surfac emoieties or receptors that are naturally present on cells of a system, e.g., a system that is a human, where the cell surface moiety or receptor provides a cell 49 entry mechanism .In various instances, the cell surfac emoiet yor receptor is cell-type specific and thus facilitates specific delivery of nucleic acids to selected cell types. id="p-151" id="p-151" id="p-151" id="p-151" id="p-151"

[0151] Nucleic acid molecules can have a large negative charge, are vulnerable for degradation in body fluids e.g.,, afte administr ration to subjects and, cannot enter a cell via simple injections or exposure to the cell. That large negative charge can be neutralized by mini- nucleosome core-proteins, including modified mini-nucleosome core proteins, to form loaded mini-nucleosomes of certain shape, size, and charge and/or that are capable of entering into cells by passive diffusio orn active transport. Various mini-nucleosome core proteins described herein allow proper binding, condensation and targeting of nucleic acids. These domains described herein, may be derived from human proteins or other organisms. One skilled in the art may contemplate modifying or engineering domains and/or mini-nucleosome core proteins described herein, e.g., with changes to the amino acid sequenc fore enhancing certain functions such as cell attachment, internalization etc. but not limited to these. One skilled in the art may also contemplate placing the domain in reverse sequenc ore by switching amino acid positions within the domain or adding various posttranslational modifications such as acetylation, glycation etc. to amino acids but not limited to these. Various mini-nucleosome core proteins described herein include at least one modified amino acid residue.

Nucleic Acid Binding Domains id="p-152" id="p-152" id="p-152" id="p-152" id="p-152"

[0152] The present disclosure includes the recognition that positively charged domains associate with nucleic acids. The present disclosure provides nucleic acid binding domains, e.g., DNA and RNA binding domains, that can be included in a mini-nucleosome core protein. In some instances, a DNA bindin gdomain present in a mini-nucleosome core protein is a DNA bindin gdomain disclosed herein. In some instance as, RNA bindin gdomain present in a mini - nucleosome core protein is a RNA binding domain disclosed herein. id="p-153" id="p-153" id="p-153" id="p-153" id="p-153"

[0153] In some particular instances, an NABD that is a DNA bindin gdomain present in a mini-nucleosom coree protein disclosed herein can be derived from a histone polypeptid e sequence. Non-viral vectors such as DNA nanoparticles utilizing poly-lysine peptides to compact DNA into smaller particle fors gene deliver (Liuy G. et al, 2003) have been used, at least some instances, with no success or significant responses in treatment of diseases (Konstan 50 M.W. et al, 2004). The present disclosure provides a significantly different approach that includes, in various embodiments, use of DNA bindin gdomain of histones, for example the amino acid sequenc KRHRKe . This amino acid sequenc servese two purpose- first it gives the highly positive charge that is needed to associate with nucleic acids, Secondly it, also gives stability to the mini-nucleosome core protein structure Third. ly, the amino acid sequenc KRHe in this NABD also is a cleavage site for proprotein convertas esthus allows efficie ntrelease of the genetic cargo in cells. id="p-154" id="p-154" id="p-154" id="p-154" id="p-154"

[0154] Other examples of NABDs are provided in Table 3. id="p-155" id="p-155" id="p-155" id="p-155" id="p-155"

[0155] A poly-arginine trac tsuch as RRRRR can be include ind a mini-nucleosome core protein to increase nucleic acid bindin gas well as to enhance positive charge and/or cell penetrat ionability of the composition. A poly-arginine trac tcan be present in a mini-nucleoso me core protein in a position suitable to facilitate penetrat ionof cells by the mini-nucleosome core protein and/or by loaded mini-nucleosomes including the mini-nucleosom coree protein. Those of skill in the art will be aware of the method ands technique thats allow determinati ofon such a position. Arginine interact withs phospholipids to form of bi- or multi-den tatehydrogen bonding from simultaneous association with the phosphates of more than one lipid head therefore interacts with the phosphate on a single lipid head group. Since, only arginine can form bi- dentate hydrogen-bonds, poly-arginines could bond with more zwitterion andic anionic lipids and therefor generatee positive curvatur alonge its contour lengt thush, resulting in negative Gaussian curvatur (Rothbae rd, J.B., et al. 2005). A poly-Argini netrac tmay also be modified to include specifical lyone or more Histidine (H) amino acid (or any other amino acid) to improve stability of the mini-nucleosome core protein. Histidine (or any other amino acid) may be inserted in any position in the poly-Arginine trac tas shown in Table 3. Other arginine-rich peptides such as ANTP Penetratin, and TAT have also shown similar impact on cell penetration. id="p-156" id="p-156" id="p-156" id="p-156" id="p-156"

[0156] The present disclosure includes the recognition that localization of a mini - nucleosome core protein to a euchromatin area of the nucleus can be facilitat byed acetylation of lysines in mini-nucleosome core proteins. The mechanism of this stabilization may be related, at least in part, to mechanisms that stabilize post-translational modifily ed histones. Methylated histones pack more tightly. Histone methylation can be dynamic. Other post translational modifications that can be applied are: phosphorylation, glycosylation, prenylation, lipoylation, 51 alkylation, acylation, glycation, nitrosylation, sulfation, carbamylation, carbonylation, sumoylation, neddylation, biotinylation, ribosylation etc. Modifications may not be limited to these mentioned here. Other modifications may include attachment of co-factors, co-enzymes, hydrophobic groups ,hydrophilic groups, smaller chemical groups ,smaller peptides etc. Such modification could also be applied to amino acids in these mini-nucleosom coree proteins described herein. Nucleic acid binding domains mentioned herein, in Table 3 can be incorporated in polypeptides at any location to enhance nucleic acid binding in combination with other domains provide din Tables 4, 5, 6, 7, 9, 10, 11 and 12.

Table 3: Exemplar y SEQ ID Name Exemplar yUtility Reference Domains NO: 1 DNA binding Enhanced DNA Bottomley domain binding M.J., 2004 RRR, RRRR, 2, 3, 4, 5 Poly-Arginines: Enhanced cell Mishra, A. et RRRRRR, (RR)X DNA binding penetration al, 2008 domain 6 Condensing John P. H.

RRLARR Enhanced DNA domain (part, of) binding; Th'ng et. al. condensation 2005 KKAKAAAKPKK Condensing Enhanced DNA John P. H. domain (part of) bindin gand Th'ng et al. condensation 2005 KKDGKKRKR 8 Condensing John P. H.

Enhanced DNA domain (part of) bindin gand Th'ng et al. condensation 2005 KKKLK 9 HTH motif (part, Enhanced DNA Uniprot of) binding KKR1RK, RKKSK 10, 11 RUNX1 binding Enhanced DNA Uniprot (part, of) binding 52 KKPKK 12 Condensing Enhanced DNA John P. H domain (part ,of) binding and Th'ng et al. condensation 2005 13 Nucleic acid Enhanced nucleic Uniprot binding acid binding and stability RHRRR 14 Nucleic acid Enhanced nucleic Uniprot binding acid binding and stability RRRRHR 15 Nucleic acid Enhanced nucleic Uniprot binding acid binding and stability KRTVRK 16 Nucleic acid Enhanced nucleic Uniprot binding acid binding ] / KRQRNR Nucleic acid Enhanced nucleic Uniprot binding acid binding 18 P53 DNA Enhanced nucleic Uniprot RVCACPGR interaction acid binding (KKK)x 19 Nucleic acid Enhanced nucleic Uniprot binding acid binding DEMGLGKT 20 Nucleic acid Nucleotide binding Uniprot binding QRE, HLSQHLN, 21, 22, 23, Nucleic acid Interaction with Uniprot KTQK, RM RVY, NRRK IFF 27 Nucleic acid RNA binding Uniprot binding RPRGRPRKHTVTS 28 Nucle icacid Enhanced nucleic Uniprot binding acid binding 53 id="p-157" id="p-157" id="p-157" id="p-157" id="p-157"

[0157] For the avoidance of doubt, the present disclosure includes modified nuclei acidc bindin gdomains, including without limitation a nucleic acid bindin gdomain of the present disclosur ine which at least one amino acid of the nucleic acid binding domain includes a modification disclosed herein.

Targeting Domains id="p-158" id="p-158" id="p-158" id="p-158" id="p-158"

[0158] Mini-nucleosome core proteins disclosed herein include targeti ngdomains that target mini-nucleosomes to one or more cells or cell types. id="p-159" id="p-159" id="p-159" id="p-159" id="p-159"

[0159] In some embodiments, a targeting domain of a mini-nucleosom coree protein is an amino acid domain that allows attachment to and enter into one or more cells or cell types. It is to be understood that targeti ngdomains can be specific to certain cell types but can also include domains that facilita teentry into cells generally. In general, a targeting domain of a mini- nucleosome core protein can contribute to one or more of attachment, cell-typ specifie cbinding, and internalizat ion.A targeti ngdomain can be, for example, a cell attachment targeti ngdomain, beta galactose bindin gdomain, fucos ebinding domain, heparin binding domain, sialic acid bindin gdomain, glycoprote bindingin domain, carbohydrate binding domain, lysophosphatidic acid binding, cAMP bindin gdomain, hyaluronan binding domain, chondroitin sulfat bindinge domain, integr inbinding domain, nucleolin bindin gdomain, collagen binding domain, clathri n bindin gdomain, Fc receptor binding domain, actin binding domain, endocytosis motif or a nuclear localization signal. In some embodiments a target, ing domain of a mini-nucleosom coree protein is an amino acid domain that allows bindin gand entry into one or more cells or cell types and that is derived from a mammal, virus, viral particle, prion, bacteria or fungal amino acid sequence. id="p-160" id="p-160" id="p-160" id="p-160" id="p-160"

[0160] For the avoidance of doubt, the present disclosure includes modified targeting domains, including without limitation a targeti ngdomain of the present disclosur ine which at least one amino acid of the targeting domain includes a modification disclosed herein.

Cel lAttachment Targeting Domains id="p-161" id="p-161" id="p-161" id="p-161" id="p-161"

[0161] Cell attachment is a means by which a mini-nucleosome core protein, or loaded mini-nucleosom includee the mini-nucleosome core protein, can adhere to cell and, in various 54 instance facilitates, entry to into the cell. Various viruses have adhesion molecules or domains that allow bindin tog host cells and enhance entry into them. For example, flu virus has hemaggluti onnin its surface that allows it to bind to sialic acid on the cell surface. The present disclosur provides,e among other things, severa lsuch domains that allow mini-nucleosome core protein binding to sialic acid, galactose, fucose, hyaluronic acid, and chondroitin sulfate, as well as glycoproteins that enhance cell attachment for internalizat ion.A mini-nucleosom coree protein disclosed herein can include one or more cell attachment targeting domains. Cell attachment targeting domains include the domains shown in Table 4. A cell attachment targeting domain of the present disclosure can be present in a mini-nucleosome core protein at any position and/or in combination with any of one or more other domains provide herein, e.g., in Tables 3, 5, 6, 7 8, 9, 10, 11 and 12.

Table 4: Exemplar yDomains SEQ ID Name Exemplary Reference NO: Utility WGREERQ 29 Cell Enhanced cell Uniprot attachment surface site on attachment via LGALS3 beta-galact ose binding NTQIH & WT4NKTPH 30, 31 CTxB domain Enhanced cell Uniprot surface attachment via galactose binding ךר TPH CTxB domain Enhanced cell Uniprot surface attachment via.

Fucose binding 55 VNRWS 33 Sialic acid Enhanced muscle Uniprot binding cell surface domain attachment via Sialic acid binding XBBBXXBX, 34, 35 Heparin Enhanced cell Cardin and ARKKAAKA binding surface Weintraub, domain attachment via 1989 Heparin binding.

QRR, SRR 36, 37 CPC motif Enhanced cell Torrent M. surface e:. al, 2012 attachment via.

Heparin binding 38 B3GAT3 WEPSRPFPVD Enhanced cell Uniprot motif surface attachment via galactose binding HRRTRKAPKRIRLPHIR 39 Herpes Enhanced cell Uniprot glycoprote in surface gD motif attachment via. glycoprotein binding KRTGQYKLGSKTGPGQK 40 Heparin Enhanced cell Uniprot binding surface domain in attachment via.

FGF2 heparin binding KKTK 41 Heparin Enhanced cell Nelson C. Di sulfate surface Paolo et al, binding 2007 attachment via 56 heparin sulfate binding domain KLRSQLVKK 42 Hyaluronan Enhanced cell Uniprot binding motif surface attachment via Hyaluronan binding RRRCGQKKK 43 Hyaluronan Enhanced cell Uniprot binding motif surface attachment via Hyaluronan binding BX(7)B 44 BX7B domain Enhanced cell Jean L. et al, 2001 surface attachment via Hyaluronan binding RIQNLLKITNLRIKFVK 45 AC 15 domain Enhanced cell Kokona surface Kouzi-K .et attachment via. al. 1989 heparin binding KKEKDIMKKTI 46 Sgl MOTIF Enhanced cell Joji I. et al, of integrin surface 1998 attachment via chondroitin sulfate binding domain HGSRFTFHRGSM, HRPH. 47, 48, Lectin bindingEnhanced cell Uniprot 49, 50, DVAR, HFNPR, WGTE surface 51 attachment via 57 Beta-galactoside binding binding domain 52 Chondroitin KKQFGAEC Enhanced cell Uniprot sulfate surface binding attachment 53 Heparan Enhanced cell RRPRPGTGPGRRPRPRPRP Uniprot sulfate surface binding attachment id="p-162" id="p-162" id="p-162" id="p-162" id="p-162"

[0162] Cell attachment can also be achieved by domains such as RGD, RODS etc.

(D’ Souza SE et al, 1991). Binding to cell surfac eproteins such as integrins, nucleolin, collagen, clathrins, Fc receptors also help viruses and other particles get entry to the cell. The present disclosur provides,e among other things, domains that allow binding to as integrins, nucleolin, collagen, clathrins, Fc receptors for increased cellul uptake.ar Cell attachment targeting domains include the domains shown in Table 5. A cell attachment targeti ngdomain provided in Table 5 can be present in a. mini-nucleosome core protein at any position and/or in combination with any of one or more other domains provide herein, e.g., in Tables 3, 4, 6, 7, 8, 9, 10, 11 and 12.

Tables: Exemplary SEQ ID Name Exemplar yUtility Reference Domains NO: KGE 54 Cell attachment Enhanced cell Maginnis motif attachmen viat M.S. et al, Integrin binding 2006 RGD, RODS 55, 56 Cel lattachment Can be used, to block D’Souza SE motif RPE transduction. et al, 1991 TTVVNPKYEGK, 57, 58 Betal integrin cell Enhanced cell Reszka A. A.

ERMSQIKRLLS attachment domain attachment via et al, 1992 Integr inbinding 58 WRHRARS 59 NS5B domain Enhanced cell Kusakawa T. attachmen viat etal, 2007 Nucleolin binding 60 GFOGER A-domains of Enhanced cell Knight C.G.

Integrins attachmen viat et al, 2000 Collage In and IV bindin tog Integrins utilm 61 ENTH domain Enhanced cell Kalthoff et al, attachmen viat 2002 Clathrin terminal domain binding WGREERQ 62 Galactose binding Enhanced cell Uniprot motif attachmen viat galactose bindin gsite on LGALS3 QSTEKRG 63 Cclec6A motif Enhanced ceil Uniprot attachment via association with Fc receptor gamma chain (FCER1G) 64 LPNTG LPXTG motif Enhanced cell Drams! et al, attachment 2008 DSPE, FQVT 65, 65 Popeye domain cAMP binding Brand, T. 2016 QSTEKRG 66 CLEC6a motif Carbohydrate Uniprot binding RQGLID 67 domain in LPAR1 Lysophosphatidic Uniprot acid binding RKKH 68 Midas motif Echo virus 1 and Pentikainen integr inbinding O. et al, 1999 59 motif. Collagen binding.

YPK, YNQYT 69, 70 Sialoadhesion Myelin associated Keim S. et al, domain glycoprotein 1994 KWNYK 71 Sialic acid binding Siglec7 Uniprot domain GPQSVKFKSPDQI 72 Adhesion domain Cytoadherence Uniprot RVGENWWY, 73, 74, Chondroitin sulfa teCell surfac e Uniprot RTLQAHHDR, 75, 76, binding attachment 77, 78, 79 RESPFSGSSR, REEIQERMR, QDSSSFHHQ, KKQFGAEC, KRALHNAEC KQKIKHVVKLK, 80, 81 Hyaluronic acid Cell surfac e Uniprot KLRCQLAKKK binding attachment id="p-163" id="p-163" id="p-163" id="p-163" id="p-163"

[0163] For the avoidance of doubt, the present disclosure includes modified cell attachment targeting domains, including withou limitt atio a ncell attachment targeting domain of the present disclosur ine which at least one amino acid of the cell attachment targeting domain includes a modification disclosed herein.

Internalizati Targeton ing Domains id="p-164" id="p-164" id="p-164" id="p-164" id="p-164"

[0164] Certain domains in viral and mammalian proteins can directly impact cellular internalizat ion.For example, domains of certain proteins and, sequential arrangement is , described in Oleson et al., 2008. For example, a PPxY-Motif is required for adenovir usentry into cells (Wodrich et al, 2010), where x could be any amino acid. Anothe exampler of an internalization targeting domain is the GTALL motif-a five-amino acid residue domain, in the carboxyl-terminus tail of leutiniz inghormone (LH) receptor direct thes ligand-recep tor complexes from a degradative to recycling pathway (Pandey, 2009). The GTALL motif also 60 shows sequenc homologye to carboxyl- terminus tetrapeptide sequenc motife DSLL, which has been suggested to participate in the internalization of P־ adrenergic receptor s.Pandey also discusses that the clathrin-dependent cargo usually contain as short sequenc motife such as YXXQ (where X could be any amino acid) ,recognize byd adaptor protein-2 (AP-2) and may contain Asn-Pro-X-Tyr sequenc (NPXe Y) motifs, which are recognized by the accessory clathri n adaptor proteins. Transferrin. NPXY moti hasf also been discusse dby Kirchhausen, 1999. NPTY is also the Endocytosis motif of APP. Anothe exampler of clathrin binding domain that allows internalization is FXDXF (where X could be any amino acid) (Lene E. Oleson. 2008).

Internalizat targetiion ngdomains include the domains provided in Table 6. id="p-165" id="p-165" id="p-165" id="p-165" id="p-165"

[0165] Other features provided by the present disclosure include one or more leucine and isoleucine residue s,which residues are highly hydrophobic in nature. In fact, leucine is the second most hydrophobic amino acid. In various embodiments leucine, residue cans serve multiple functions in the composition of mini-nucleosome core proteins. First, the hydrophobicity of the nonpol arface of an amphipathic molecule plays an important role in stabilizing the peptide secondary structur (Chene Y. et al, 2007). Secondly dileuci, ne-type of signal motifs have been shown to be essential for internalizatio andn trafficking of membrane receptors and membrane proteins into subcellula comparr tments. For example, GLUT4 (glucose transporte 4),r LDL (low density lipoprotein); LH (leutinizing hormone) ,TGN (Trans-Golgi network) all have dileucine motifs that help internalization into cells. Fc receptor dileucine motif also signals for endocytosis (Wu Z. and Simister N.E., 2001). An internalization targeting domain provided in Table 4 can be present in a. mini-nucleosom coree protein at any position and/or in combination with any of one or more other domains provide herein, e.g., in Tables 3, 4, , 7, 8, 9, 10, 11 and 12.

Table 6: Exemplar yDomains SEQ Exemplary Name Reference ID Utility NO: 61 FXDXF 82 FXDXF- motif Clathrin binding Lene E. Oleson motif facilitates JBC. 2008 internalization PPSY 83 PPxY-Motif Facilitates H Wodrich et al, 2010 Adenovir us Entry. At the end of the sequence. fednfvp 84 7-mer peptide from Enhanced Lene E. Oleson amphiphysin. InternalizationJBC. 2008 85, 86, Internalization YIRV, YADW, YTQV Enhanced Zrarate et al, 2007 87 motif Internalization KKRPKP 88 Prion (Sunyach, 2003).

Is sufficient to internalizat ion direct motif internalization.

SSDDE, RRASS 89, 90 CcN motif Efficient nuclear (David A Jans. transport and 1995 JBC) localization (YXXL)2 91 Internalization For viral entry Inabe K et al, and 1999 motif of bovine leukem virusia incorporation of viral envelope protein into virions.

LPLTG, LAFTG 92, 93 Sorting signal Sortase Ton-That EL,, and dependent entry. O.

Schneewind. 2003 L L LI, IL 94, 95, Leucines, Increase d Chen Y. et al, 96, 97 2007 Isoleucine hydrophobicity 62 for polypeptid e stability LL 98 Dileucine Enhanced WuZ. and cellular Simister N.E., internalization2001 99 Heparan sulfate KRRHPKK Cardin-Weintra ub Uniprot motif binding EPS, EPNLPEE, ND 100, Mannose binding Enhanced Uniprot 101, domian cellular 102 internalization NFR 103 N-acetyl-D- Enhanced Uniprot cellular glucosamine binding internalization YWV 104 PDZ binding Enhanced Uniprot cellular internalization AICKRIPNKKPGKRT 105 Heparin binding Enhanced Uniprot cellular internalization VAR, KIL 106 Receptor binding Enhanced Uniprot (CXCL12) cellular internalization RCPCR, 107, Heparin binding Enhanced Uniprot RANVKHLKILN, 108, cellular VARLKNNNRQV 109 internalization VRKKP, no, PDGFA bindin tog Enhanced Uniprot YVRKKPKLK 111 its receptor cellular internalization 63 ISRRLI 112 PDGFB binding to Enhanced Uniprot its receptor cellular internalization 113, Gag binding LTKRSRQ, Enhanced Uniprot NRKISVQRL cellular 114 internalization YYKQRLI 115 Nucleocytoplasmic Enhanced Uniprot transport cellular internalization id="p-166" id="p-166" id="p-166" id="p-166" id="p-166"

[0166] For the avoidance of doubt, the present disclosure includes modified internalization targeting domains, including withou limitatt ion an internalization targeting domain of the present disclosure in which at least one amino acid of the internalization targeting domain includes a modification disclosed herein.

Nucleus Targeting Domains id="p-167" id="p-167" id="p-167" id="p-167" id="p-167"

[0167] In various embodiments, it is important that follow, ing cellul entry,ar a nucleic acid cargo reaches the nucleus. Nuclear internalization signal ors bindin tog the nuclear import machinery are key to nuclea localr ization. Functional eukaryotic nucle arlocalization signals are widespread in terminal proteins of bacteriophages (Redrej o-Rodriguez et. al, 2012). Chan and Jans have shown that polylysine by itself doesn’t function as a nucle arlocalization signal. Thus, adding a nuclea targetir ngsignal tos enhance non-viral gene transf eris a logical approach (Chan and Jans, 1999). Location of NLS in the polypeptide is also key for its function. We have listed the NLS sequences in Table 7 for enhanced nuclea entryr and provide dcertain preferred locations of NLS signal within mini-nucleosome core proteins for efficie ntnuclea entryr in Table 13. Domains mentioned herein, in Table 7 can be incorporated in mini-nucleosom coree protein at any location to enhance nucleic acid bindin gin combination with other domains provided, in Table 3, 4, 5, 6, 8, 9, 10, 11 and 12.

Table: 7 64 Exemplar yDomains SEQ Source Exemplary Reference ID protein Utility NO: KKKYKLK 116 Nuclear Gag pol Uniprot localization signal KKRKLE 117 LMNA Nuclea r Uniprot localizati on signal TRSK 118 VP22 Nuclear Uniprot localization signal HRKRKR 119 Aprataxin Nuclear Uniprot localizati on signal NKRKRK 120 SAP30L Nuclear Uniprot localization signal AEKSKKK 121 HMGBI Nuclear Uniprot localization signal RKSK, KRVK 122, HIPK2 Nuclea r Uniprot 123 localizati on signal KRK 124 NFATCI Nuclear Uniprot localization signal 125 NFKB Nuclear LQQTPLHLAVI Uniprot inhibitor localization alpha 65 signal contains ankyrin repeats RRPR, PRPR, RPPP 126, Bovine Nuclear Uniprot 127, localization Herpe s 128 Virus signal for bovine herpes.

RKKRKGK 129 DAG1 (dystroglycan). Uniprot In the c- terminal PAAKRVKLD 130 c-Myc Nuclear Uniprot localization signal 131 Nuclea r KLKIKRPVK TUS Uniprot localizati on signal PKKKRKV 132 SV40 Nuclear Uniprot localization signal QRKRQK 133 NFKB Nuclear Uniprot localization signal KRPR 134 TOPBP 1 Nuclea r Uniprot localizati on signal RKRRRP 135 DEDD2 Nuclear Uniprot localization signal 136 Nuclea r KKGRRNRFK I INF 1A Uniprot localizati on signal 66 Attorney Docke tNo.: 2013247-0013 137 Nuclear Uniprot RIIRDRLNTELDRLASLLPFPQDVINKLDK AFIR localizati on signal KRGRKP 138 CBX2 Nuclea r Uniprot localization signal KKRAGRRIFKETR 139 DREBE1 Nuclea r Uniprot localization signal id="p-168" id="p-168" id="p-168" id="p-168" id="p-168"

[0168] For the avoidance of doubt, the present disclosure includes modified nucle us targeting domains including, without limitation a nucleus targeting domain of the present disclosure in which at least one amino acid of the nucle ustargeti ngdomain includes a modification disclosed herein.

Cell-type Specifi cTargeting Domains id="p-169" id="p-169" id="p-169" id="p-169" id="p-169"

[0169] In various embodiments, it is most desirable that large concer ntration of the particles home into the desired cel ltype. This allows for increased uptake and increased expression- two favorable gene therapy output. In literatur theree, are very few motifs that have been discovered for such properties. Most of thes ecome from experiment thats have shown viral tropism to be different from different capsids. The present disclosure includes in, various embodiments use, of some of those defined motifs to, enhance expression in neurons, muscles , liver, lung, kidney, endothel cellial ors tumor sites .Cell-type specific targeting domains includ e the domains shown in Table 8. A cell-type specific targeting domain of Table 8 can be present in a mini-nucleosome core protein at any position and/o rin combination with any of one or more other motif provides herein, e.g., in Tables 3, 4, 5, 6, 7, 9, 10, 11 and 12.

Table :8 SEQ ID Exemplary Utility Exemplary Domains Reference NO: 67 ASSLNIA 140 Muscle targeting Yu C-Y. et al. 2009 SKTFNTHPQSTP 141 Muscle targeting Y Seow et al. 2010 YKQCHKKGGHCFPKEK 142 Muscle targeting Uniprot LGKMDCRWKWKCCKKGSG 143 Muscle targeting Uniprot HGSRFTFHRGSM 144 Muscle targeting Uniprot KKEEEKKEEEKKEEE 145 Rena ltargeting Wischnjow A, et al, 2016 LIFHKEQ 146 IJ V M< targeting Uniprot KFNKPFVFLI 147 Lung targeting Bearing H. et al, 2003 QPEHSST 148 Endothelia celll targeting Work, L. M. et. al, 2006 EYHHYNK 149 Vascula rsmooth muscle ceil Work, L. M, et. al, targeting 2004 150 Tumor homing zkrap W, et. al, 1998 NGR GEKGEP 151 Facilitate phagocytosis by Uniprot monocytes KTKKK, KALKKK, KGKKK 152, 153, Phagocytosis of the particles. Caberoy N.B. et al, 154 2010 CSVTCG 155 Interacti onwith CD36; bind to Asch A.S., et. al 1992 cancerous cells.

LRE 156 Neuron targeting by enhance d Hunter D. D. et al, neurona attachmenl t. 1989 YKYNLNGRES 157 Lung targeting Asokan A, et ai, 2006 YRSL 158 Basolateral targeting Anderson E., et al, 2005 KGGK7 159 Actin-binding Dahlin-Huppe K. et al., 1997 KKKQYTSIHHG 160 Basolateral sorting Zheng P. et al, 1998 68 KDEL 161 Endosomal Reticulum targeting Chinnapen D.J. et al, 2007 LADQDYTKTA 162 Retrograde transport Tervo D.G.R , et. al, 2016 DDNN 163 Corin surface targeting Uniprot SAVTTVVN 164 ITGB1 interaction with Uniprot ITGB1BP1 id="p-170" id="p-170" id="p-170" id="p-170" id="p-170"

[0170] For the avoidance of doubt, the present disclosure includes modified cell-type specific targeting domains includ, ing without limitati aon cell-type specific targeting domain of the present disclosure in which at leas tone amino acid of the cell-type specific targeting domain includes a modification disclosed herein.

Nucle icAdd Release Domains id="p-171" id="p-171" id="p-171" id="p-171" id="p-171"

[0171] In some embodiments a ",nucleic acid release domain" ("NARD") of a mini- nucleosome is an amino acid domain that causes or facilitat relees as eof a nucleic acid cargo of a loade dmini-nucleosome (e.g., release of one or more of the nuclei acidc s associate dwith a mini- nucleosome core protein of a loaded mini-nucleosome) In .various embodimen ts,by controlling or regulati theng conditions under which a nuclei acidc cargo is released, a nucleic acid release domain can improve delivery a nucle icacid cargo to cells, e.g., to the cytoplasm or nucle usof cells. id="p-172" id="p-172" id="p-172" id="p-172" id="p-172"

[0172] It is highl ydesirable that nucleic acids associated with a loade dmini-nucleosome core protein (e.g., when delivered to a subject or system) do not releas efrom a loaded mini- nucleosome prior to the loaded mini-nucleosom entere ing a cell (e.g., a cel lof the subject or system) Withi. n a cell rele, ase of nucleic acid cargo in the cytoplasm or nucle usmay be preferred. Various protease ands endopeptida knownses in the art could cause or facilitat relee ase of one or more nuclei acic ds of a loaded mini-nucleosome inside cells, e.g., causing or facilitating disassociation of one or more nuclei acidsc of a loade dmini-nucleosome from the mini-nucleosom coree protein of the loaded mini-nucleosome. Proprotein convertas esand endopeptidase ares exemplary agents that cleave polypeptides at certain amino acid domains, 69 which phenomeno isn utilized herein to provide mini-nucleosome core proteins that can release an associated nucleic acid cargo upon delivery into a cell (e.g., a cell of a subject or system) e.g.,, into the cytoplasm or nucleus of a cell. id="p-173" id="p-173" id="p-173" id="p-173" id="p-173"

[0173] KRH is an exemplar cleavagy domaine that can be included in a mini- nucleosome core protein as a nucleic acid release domain. KRH is the cleavage site for Pcskl and Pcsk2. To provide one non-limiting example of a KRH cleavage site, proglucagon is post- translationally processed in a tissue-specif manneric in pancreati Ac cells and intestinal cells by Pcskl 0rPcsk2. id="p-174" id="p-174" id="p-174" id="p-174" id="p-174"

[0174] NRRKKRAL is an exemplary cleavage domain that can be include ind a mini- nucleosome core protein as a nucleic acid release domain. To provide one non-limit ingexample of an NRRKKRAL cleavage domain, NRRKKRAL is a Turin cleavage site of for TGFB1.

Anothe exemplarr ycleavag domaine is KSVKKRSVSEIQ, which is a Turin cleavage site in parathyroid hormone. id="p-175" id="p-175" id="p-175" id="p-175" id="p-175"

[0175] Various other cleavage domains are known in the art and can be included in a mini-nucleosom coree protein as a nucleic acid release domain. As those of skill in the art will appreciate cleavag, sitese can also be predicted in silico using bioinformatics platforms such as Expasy, OmicX, PROSPEROUS, Propl.O, SignalP-5.0, MEROPS, CutDB, Peptide Cutt eretc. id="p-176" id="p-176" id="p-176" id="p-176" id="p-176"

[0176] The present disclosure provides that cleavage domains of the present disclosure can be included in mini-nucleosom coree proteins to cause or facilita tereleas eof a nucleic acid cargo of a loaded mini-nucleosomes in cells (e.g., cells of a subject or system) e.g.,, in cytoplasm or nucleus. Domains provided herein, including those provided in Table 9, can be present in a mini-nucleosome core protein of the present disclosure (e.g., a modified mini-nucleosome core protein) at any position within the mini-nucleosome core protein. Tor the avoidance of doubt, a nucleic acid release domain of the present disclosure can be present in a mini-nucleosome core protein in combination with other domains provided herein, including withou limitationt those provided in Tables 3, 4, 5, 6, 7, 8, 10, 11 and 12. When present anywhere within a mini - nucleosome core protein of the present disclosure, a nucleic acid release domain of the present disclosur cane enhance release of a nucleic acid cargo of a loaded mini-nucleosome.

Table: 9 70 Exemplar yDomains SEQ ID Exemplar yUtility Reference NO: GRKKRRQRRRPQ 165 Release at extracellula or r Tian and Huang intracellular sites depending on et al, 2011 tissues expressing furin. 166 Release at extracellular or KRH Uniprot intracellular sites depending on tissues expressing Pcskl and Pcsk2 KSVKKRSVSEIQ 167 Release at extracellula or r Uniprot intracellular sites depending on tissues expressing Pcskl and Pcsk2 168 Release at extracellula or r Tian and Huang NRRKKRAL intracellular sites depending on et al, 2011 tissues expressing furin.

KFERQ 169 Breakdown in the lysosomes. Park J.S. et al., 2016 VRGP 170 Cleavage by Thrombin Uniprot 171 Cleavage by Plasmin Uniprot NKDS, NRDN ANNR 172 Cleavage by Hementin Uniprot HL 173 Cleavage by MMP9 Uniprot RI, EI, GQ, RS, RD, 174, 175, Cleavage by autolysis Uniprot RN, R( RG, RL DA, 176, 177, 178, 179, RA, GS, LT, FS, GL SA, DP, GT, GC, RQ, 180, 181, LS, HA 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 71 192, 19.3, 194, 195 FV, QH, EA, AL, LY, 196, 197, Cleavage by Pepsin Uniprot YL, GF, PS, RE, DP, 198, 199, 200, 201, PL QS 202, 203, 204, 205, 206, 207 ND 208 Cleavage by BMP 1 Uniprot id="p-177" id="p-177" id="p-177" id="p-177" id="p-177"

[0177] For the avoidance of doubt, the present disclosure includes modified nuclei acidc releas edomains, including without limitation a nucleic acid release domain of the present disclosur ine which at least one amino acid of the nucleic acid release domain includes a modification disclosed herein.

Stability Domains id="p-178" id="p-178" id="p-178" id="p-178" id="p-178"

[0178] In some embodiments, a "stability domain" of a mini-nucleosome is an amino acid domain that allows loaded mini-nucleosomes to stay stable in bodily fluids, cytoplasm and the nucleus. id="p-179" id="p-179" id="p-179" id="p-179" id="p-179"

[0179] Particle stability is important for safe passage into cells and longevity of expression. There are several reasons for particles to lose stability. First, particle shoulds be stable in blood and other bodily fluid s.Secondly particle, needs to safely traverse the endosomal entry and escape safely to make it out to the cytoplasm. Viral particle ors recycled receptors use several domains to enter the endosome and escape it. We provide examples of mini-nucleosome core proteins that incorporate endosomal entry and escape domains to increase stability.

Domains mentioned herein, in Table 10 can be incorporated in mini-nucleosome core protein preferabl aty the C- terminal but also at any location to enhance stability of the mini-nucleosome core protein when combine dwith other domains provide din Table 3, 4, 5, 6, 7, 8, 9, 11 and 12.

One skilled in the art may also contemplate fluorination of hydrophobic amino acids in the peptides to provide means of increasin gprotein stability, enhanced assembly etc. and to 72 strengthen ligand-recep intertor actions. One skilled in the art may also contemplate other post translational modifications to amino acids in the peptides to provide means of increasin gprotein stability, enhanced assembly etc. and to strengthen ligand-receptor interactions.

Table: 10 SEQ ID Exemplar yUtility Exemplary Domains Reference NO: YTRF 209 Endocytos issignal for Pande yK.N.

Transferrin receptor 2009 GDAY 210 Internalizat signalion for Pande yK.N. endocytosis of NPRA 2009 LLEE 211 Endosomal entry of Cd209 Uniprot RKKRRQRRR 212 Najjar K, et.

Allows for endosomal escape al., 2015 YKSL 213 Endosomal entry of Cd209 Uniprot YENF 214 Endosoma lentry of CELClOa Uniprot FQDL 215 Endosoma lentry of CELClOa Uniprot 216 Integrin conjugation, YIGSR Graf, J et al, increased cell attachment 1987 IKVAV 217 Cel lMembrane Penetrating Tashiro, K, et Peptide, cell attachment al 1989 EFAKFE 218 Recycling endosomes Uniprot LLEEEQLRGLGFRQTRGYKSL 219 Endosomal entry of Cd209 Uniprot id="p-180" id="p-180" id="p-180" id="p-180" id="p-180"

[0180] For the avoidance of doubt, the present disclosure includes modified nuclei acidc releas edomains, including without limitation a nucleic acid release domain of the present disclosur ine which at least one amino acid of the nucleic acid release domain includes a modification disclosed herein. 73 Oligomerization domains id="p-181" id="p-181" id="p-181" id="p-181" id="p-181"

[0181] Oligomerization is a chemical process by which monomer sassociate to form multimers, including dimers and higher order macromolecular complexes. Oligomerization of proteinaceous molecules is often facilitated by domains that promote association of monomers. id="p-182" id="p-182" id="p-182" id="p-182" id="p-182"

[0182] In some embodiments, an "oligomerization domain’'’ of a mini-nucleosome is an amino acid domain that allows mini-nucleosome core proteins or loaded mini-nucleosomes to associate in higher order structur essuch as homodimer, heterodimer tetr, amer, octamers or other higher order structures. Oligomerization can reduce the size of a loaded mini-nucleosome. A multimer ofs mini-nucleosom coree proteins can include two or more of the same mini - nucleosome core protein (e.g., two mini-nucleosome core proteins having the same amino acid sequence and/or) can include two more distinct mini-nucleosome core proteins (e.g., two mini- nucleosome core proteins having different amino acid sequences) Examples. of oligomerization domains provided herein are not in any way limiting and one skilled in the art. can appreciated that such domains may be recognized or identified by various methods including yeast-two hybrid screenin g,affini typurification coupled to mass spectrometr textmining,y, or by application of artificial intelligenc ande machine learning One. skilled in the art can also create an inducible system of forming loaded, mini-nucleosomes using an inducible homodimerization system and/or chemically induced dimerization. id="p-183" id="p-183" id="p-183" id="p-183" id="p-183"

[0183] In some embodiments, an oligomerization domain can include 3 or more amino acids. Oligomerization domains disclosed herein, e.g., in Table 11, can be incorporated in mini - nucleosome core protein at any position of a mini-nucleosom coree protein, e.g., in combination with other domains provided herein, e.g., in Table 3, 4, 5, 6, 7, 8, 9,10 and 12. In certai n particular embodiments, an oligomerization domain is positioned at the C- terminus of a mini- nucleosome core protein.

Table: 11 Exemplar yDomains SEQ ID Exemplar yUtility׳ Reference NO: 74 LIRERTE 220 Dimerization Tucker C.L., et al, 1999 LVTYRTQ 221 Dimerization Tucker C.L., et al, 1999 IITFTK 222 Human PTB Domain helps Markovtsov, V et dimerization al, 2000 HJ XK 223 Human PTB Domain helps Markovtsov, V et dimerization al, 2000 PIRTLSK 224 Human PTB Domain helps Markovtsov, V et dimerization al, 2000 YGNSPLHRFK 225 Human PTB Domain helps Markovtsov, V et dimerization al, 2000 FFQKDR 226 Human PTB Domain helps Markovtsov, V et dimerization al, 2000 KSRP 227 Human PTB Domain helps Markovtsov, V et dimerization al, 2000 YVM 228 GRB2 domain mediated, Uniprot interaction YMKM 229 YXXL domain helps Uniprot oligomerization RSSSFG 230 Protein-protein interaction Uniprot LKIRGRER, 231, 232 P53 oligomerization (part of) Uniprot LKIRGRKR HVIFKKVSR 233 Heterodimerizat ofion SAG with Uniprot Rho RGPRV 234 Polymerization of Fibrin Uniprot RANVKHLK 235 Polymerization of CXCL12 Uniprot YPKAG, YPRTG 236, 237 Dimerization of DPP-IV Tang, H-K et. al, 2011 75 id="p-184" id="p-184" id="p-184" id="p-184" id="p-184"

[0184] For the avoidance of doubt, the present disclosure includes modified oligomerization domains, including without limitation an oligomerization domain of the present disclosur ine which at least one amino acid of the oligomerizati ondomain includes a modification disclosed herein.

Linkers id="p-185" id="p-185" id="p-185" id="p-185" id="p-185"

[0185] It is known in the art of creating fusion proteins that proteins can, some instances, benefit from inclusion of a linker. The present disclosure includes mini-nucleosome core proteins that include one or more linker e.g.,s, between two domains of a mini-nucleosome core protein. Linkers can contribute to protein structure stability. In some cases, linkers work as a separation between domains and in others they can directl affy ect function of proteins. Some linkers increase stiffness thus allowing effective separation of protein domains. Linkers also may be implemented to introduce cleavage sites. Linkers have been used for these reasons in the fiel d of protein engineering. However ,in the context of non-viral gene transf erthis strategy hasn’t been utilize Wed. show here that linkers can be successfull usedy to engine domainser for functional purposes such as selecti vetransduction, gene delivery and transgene expression in desired cell types (Figur e10). In some cases, linkers separate domains and those of skill in the art will appreciate that non-functiona aminol acids between functional domains have been referred to in the art as spacers. For the avoidance of doubt, the term linker as used herein includes spacers. id="p-186" id="p-186" id="p-186" id="p-186" id="p-186"

[0186] In some embodiments, a linker sequence can include 1 or more amino acids.

Linker amino acid sequences disclosed herein, e.g., in Table 12, can be incorporated in mini- nucleosome core protein between domains as shown in SEQ ID NOS: 238-335, where a linker could be a linker having any of the amino acids or amino acid sequences provide din Table 1 and 12. The linkers may contain other amino acid sequences not limited to those provided in Table 12. Linker sequences may also be generated via program calle LINKd ER, which searches databas eof linker sequences using user-chosen inputs and generate output of linker sequences that fit the criteria .Threonine, serine, glycine, proline, arginine and alanine are preferred residues in natural linkers and thus, in mini-nucleosome core proteins.

Table 12.

Linkers SEQ ID NO: L 238 LL 239 GSS 240 241 GSSGSS 242 GGS sss 243 ssssss 244■ 245 GGSGG 246 GGSGGGGG GGS GGHMGS GG 247 A(EAAAK)nA 248 (AP)n 249 (KP)n 250 (EP)g 251 GT 252 AAGAATAA 253 GSGSGSGS 254 GGSSG 255 PP 256 ww 257 MH 258 QP 259 PL 260 CM 261 RM 262 RK 263 QR 264 77 HR 265 FW 266 PW 267 HR 268 DH 269 QS 270 WG 271 7?2 GM KP 273 LF 274 YQ 275 RI 276 FY 277 FN 278 TA 279 HY 280 QV 281 282 DW AW 283 YI 284 HT 285 CH 286 HP 287 TA 288 EM 289 KH 290 ML 291 AQ 292 YL 293 78 FI 294 KY 295 WR 296 LA 297 FS 298 AR 299 FN 300 ET 301 LW 302 NE 303 LH 304 MH 305 FY 306 PH 307 YE 308 HK 309 PW 310 HF 311 IM 312 DH 313 VH 314 DR 315 RI 316 317 QS 318 FC GM 319 HR 320 HN 321 EC 322 VT 323 TH 324 CR 325 FQ 326 EV 327 kt 328 TD 329 330 SF ST 331 QV 332 YK 333 NQ 334 QK 335 id="p-187" id="p-187" id="p-187" id="p-187" id="p-187"

[0187] For the avoidance of doubt, the present disclosure includes a modified linker, including without limitation a linker of the present disclosure in which at least one amino acid of the linker includes a modification disclosed herein.

Mini-nucleosome core proteins id="p-188" id="p-188" id="p-188" id="p-188" id="p-188"

[0188] A mini-nucleosome core protein can include one or more domains provide d herein. id="p-189" id="p-189" id="p-189" id="p-189" id="p-189"

[0189] Mini-nucleosome proteins disclosed herein include at least a. positively charged amino acid sequenc thate contain as nucleic acid binding domain, a targeting domain and/or a nucleic acid release domain and/or a stability domain. The mini-nucleosom coree protein can be sequences that have e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a mini-nucleoso mecore protein as set fort hin any of SEQ ID NOs: 336-388. In various embodiments, a mini-nucleosome core protein has at least 65%, at least ד0%י at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 80 98%, at least 990؟׳, or 100% sequenc identitye with a mini-nucleosome core protein as set forth in one of SEQ ID NOs: 388-394, 399, 401, or 447. id="p-190" id="p-190" id="p-190" id="p-190" id="p-190"

[0190] In some embodiments, a. mini-nucleosome core protein may contain amino acid sequenc lengthe from 10 to 100 amino acids. Amino acids, e.g., 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 55, 70, 75, 80, 85, 90, 95, or 100 amino acids. In certain embodiments, a. mini- nucleosome core protein can have a length of, e.g., 15 to 90 amino acids, 20 to 80 amino acids, to 70 amino acids, 20 to 60 amino acids, or 30 to 40 amino acids. id="p-191" id="p-191" id="p-191" id="p-191" id="p-191"

[0191] In certain embodiments, a mini-nucleosome core protein includes one or more domains disclose dherein and one or more amino acids that is not present in a domain disclosed herein. In certain instances, amino acids not present in a domain disclosed herein that are N- terminal or C-terminal of a domain disclosed herein can be referred to as "flanking amino acids", and the sum of all amino acids present in a. mini-nucleosome not present, in any domain disclosed herein can be referred to as the "non-domain amino acids." id="p-192" id="p-192" id="p-192" id="p-192" id="p-192"

[0192] In various embodiments, non-domain amino acids of a mini-nucleosom coree protein can have a sequence that contributes to the charge of the mini-nucleosom coree protein.

In various embodiments, non-domain amino acids of a mini-nucleosome core protein include at least 10% positively charged amino acids, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% positively charged amino acids. id="p-193" id="p-193" id="p-193" id="p-193" id="p-193"

[0193] In some embodiments, at pH7, a mini-nucleosome core protein may have a total positive charge in between 10 and 100. id="p-194" id="p-194" id="p-194" id="p-194" id="p-194"

[0194] In some embodiments, a mini-nucleosome core protein can contain one or more nucleic acid binding domains placed at any location of the amino acid sequence. In some cases, the mini-nucleosome core protein may contain only the nucleic acid binding domains. In some cases, the mini-nucleosom coree protein may contain on the nucl eic acid binding domains and the poly-Argini nedomains .In some cases, the mini-nucleosome core protein may contain on the nucleic acid binding domains and the targeti ngdomains. In some cases, the mini-nucleoso me core protein may contain only the poly-Arginine domains and the targeting domains. In some cases, the mini-nucleosom coree protein may contain only the poly-Arginine domains, nucleic acid releas edomains and the targeting domains. 81 id="p-195" id="p-195" id="p-195" id="p-195" id="p-195"

[0195] In some embodiments, a mini-nucleosome core protein may contain one or more poly-Arginines placed at any location of the amino acid sequence. The poly-Arginine sequence may contain 4-30 Arginines. id="p-196" id="p-196" id="p-196" id="p-196" id="p-196"

[0196] In some embodiments, a mini-nucleosome core protein may contain one or more targeting domains. The targeting domain may be place dat any location in the amino acid sequenc ofe the mini-nucleosome core protein. id="p-197" id="p-197" id="p-197" id="p-197" id="p-197"

[0197] In some embodiments, a. mini-nucleosome core protein may contain one or more nucleic acid release domains. Preferably, the nuclei acidc release domains are placed in the mi ddle of the amino acid sequenc ofe the mini-nucleosome core protein. Preferably the, nucleic acid releas edomains are placed after 6 amino acids from the N-terminus or before 6 amino acids from the C-terminus. id="p-198" id="p-198" id="p-198" id="p-198" id="p-198"

[0198] In some embodiments, a. mini-nucleosome core protein can contain one or more stability domains. Preferably, the stability domains are placed in the C-terminal of the amino acid sequenc ofe the mini-nucleosome core protein. In some cases, the stability domains are placed in the N-terminal of the amino acid sequenc ofe the mini-nucleosome core protein. id="p-199" id="p-199" id="p-199" id="p-199" id="p-199"

[0199] In some embodiments, a. mini-nucleosome core protein can include one or more oligomerization domains. In certain particular embodiments, the oligomerization domains are positioned at the C-terminus of the amino acid sequenc ofe a mini-nucleosome core protein. In some cases, the oligomerization domain is positioned at the N-terminus of the amino acid sequenc ofe a mini-nucleosome core protein. id="p-200" id="p-200" id="p-200" id="p-200" id="p-200"

[0200] Thus, for the avoidance of doubt, a mini-nucleosom coree protein, as set forth herein, can include (a) a nuclei acidc bindin gdomain (NABD), and. (b) a targeting domain, and in some embodiments can include (a) a nucleic acid binding domain (NABD), (b) a targeting domain, and (c) a nucle icacid releas edomain. Those of skill in the art will appreciate from the present disclosure that a polypeptide including these component wills constitute a mini - nucleosome core protein as disclosed herein, optional lysubjec tto additional limitations set fort h herein and/or including, without limitation, one or more further domains provided herein or otherwise known in the art. In some embodiments, a mini-nucleosom coree protein can include a nucleic acid binding domain having at least 65% sequenc identie ty'with a nucleic acid binding domain as set forth in any of SEQ ID NOs: 1-28 (e.g., as set forth in Table 3), e.g., at least 65%, 82 at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or that differs train a nucleic acid bindin gdomain as set forth in any of SEQ ID NOs: 1-28 by no more than two amino acid changes (e.g., a deleti on,addition, or substitution, e.g., a. conservative substitution) or no more than one amino acid changes. In some embodiments a mini-, nucleosome core protein can include a. targeting domain that is a. cell attachment targeting domain having at least 65% sequenc identitye with a cell attachment targeting domain as set forth in any of SEQ ID NOs: 29-53 (e.g., as set forth in Table 4), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%. at least 90%, at least 95%, and/or that differs from a cell attachment targeting domain as set forth in any of SEQ ID NOs: 29-53 by no more than two amino acid changes (e.g., a deletion, addition, or substitution, e.g., a. conservative substitution) or no more than one amino acid changes. In some embodiments, a mini-nucleosom coree protein can include a targeting domain that is a cell attachment targeting domain having at least 65% sequenc identite withy a cell attachment targeti ngdomain as set fort hin any of SEQ ID NOs: 54- 81 (e.g., as set forth in Table 5), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%0, at least %)%, at least 95%, and/or that differs from a cell attachment targeting domain as set forth in any of SEQ ID NOs: 54-81 by no more than two amino acid changes (e.g., a deleti on,addition, or substitution, e.g., a conservative substituti on)or no more than one amino acid, changes. In some embodiments a mini-nucleosome, core protein can include a targeting domain that is an internalization targeting domain having at least 65% sequenc identite withy an internalization targeting domain as set forth in any of SEQ ID NOs: 82-115 (e.g., as set forth in Table 6), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or that differs from an internalization targeting domain as set fort hin any of SEQ ID NOs: 82-115 by no more than two amino acid changes (e.g., a deleti on,addition, or substitution, e.g., a conservative substituti on)or no more than one amino acid changes. In some embodiments, a. mini-nucleosom coree protein can include a. targeti ngdomain that is a. nucleus targeting domain having at least 65% sequenc identitye with a nucleus targeti ngdomain as set forth in any of SEQ ID NOs: 116-139 (e.g., as set forth in Table 7), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or that differs from a nucleus targeting domain as set fort hin any of SEQ ID NOs: 116-139 by no more than two amino acid changes (e.g., a deletion, addition, or substitution, e.g., a conservative substitution) or 83 no more than one amino acid changes. In some embodiments, a mini-nucleosom coree protein can include a targeting domain that is a cell-type specific targeting domain having at least 65% sequenc identie withty a. cell-type specifi ctargeting domain as set forth in any of SEQ ID NOs: 140-164 (e.g., as set forth in Table 8), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or that differs from a cell-type specific targeting domain as set fort hin any of SEQ ID NOs: 140-164 by no more than two amino acid changes (e.g., a deleti on,addition, or substitution, e.g., a conservative substituti on)or no more than one amino acid changes. In some embodiments, a mini-nucleosom coree protein can include a nucleic acid release domain having at least 65% sequenc identite withy a nucleic acid release domain as set fort hin any of SEQ ID NOs: 165-208 (e.g., as set forth in Table 9), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or that differs from a nucleic acid releas edomain as set forth in any of SEQ ID NOs: 165-208 by no more than two amino acid changes (e.g., a deleti on,addition, or substitution, e.g., a conservative substitution) or no more than one amino acid changes. In some embodiments a mini-, nucleosome core protein can include a stability domain having at least 65% sequenc identite y with a stability domain as set forth in any of SEQ ID NOs: 209-219 (e.g., as set forth in Table ), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or that differs from a stability domain as set forth in any of SEQ ID NOs: 209-219 by no more than two amino acid changes (e.g., a deleti on,addition, or substitution, e.g., a conservative substitution) or no more than one amino acid changes. In some embodiments, a mini-nucleosom coree protein can include an oligomerizati ondomain having at least 65% sequenc identite withy an oligomerization domain as set forth in any of SEQ ID NOs: 220-237 (e.g., as set forth in Table 11), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or that differs from an oligomerizati ondomain as set forth in any of SEQ ID NOs: 220-237 by no more than two amino acid changes (e.g., a. deleti on, addition, or substitution, e.g., a conservativ substie tuti on)or no more than one amino acid changes. In some embodiments, a mini-nucleosome core protein can include a linker domain having at least 65% sequenc identitye with a. linker domain as set forth in any of SEQ ID NOs: 238-335 (e.g., as set forth in Table 12), e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and/or that differs from a linker domain as set 84 forth in any of SEQ ID NOs: 238-335 by no more than two amino acid changes (e.g., a deleti on, addition, or substitution, e.g., a conservative substituti on)or no more than one amino acid changes. id="p-201" id="p-201" id="p-201" id="p-201" id="p-201"

[0201] Those of skill in the art that domains of a mini-nucleosom coree protein provided herein can be arranged in any order ,orientation, or sequenc ase provided herein or as will otherwise be understood from the present disclosure by those of skill in the art. For instance, those of skill in the art will appreciate the intended use of linkers, e.g., as optional sequences that can be included individually or in a tandem plurality betwee anyn pair of domains or adjacent to any domain, with or without one or more intervening amino acids not specifical lydisclosed herein. Thus, for example, a NABD can be C-terminal or N-termina ofl a. targeting domain.

Additional domains provided herein, including without limitation addition Nal ABDs or additional targeting domains, can be C-terminal or N-terminal of NABD and C-termina orl N- terminal of a targeti ngdomain. Moreover ,for each domain present in mini-nucleosom coree protein, including a linker, one or more linker domains can be included C-terminal of the domain or N-termina ofl the domain. Exemplar׳ ymini-nucleosom proteinse are provide dherein. As wall be readily apparent to those of skill in the art from the present disclosur e,domains provided herein are modular and can be include withd their intende functiond in any order and/or thereby provide the mini-nucleosome with the intende utilityd or functionality regardless of the order in which they are present. id="p-202" id="p-202" id="p-202" id="p-202" id="p-202"

[0202] Those of skill in the art will furthe appreciater that mini-nucleosome core proteins of the present disclosure can include any numbe orr type of modifications (e.g., posttranslational modifications) known in the art. Such modifications include, without limitati on,pegylation, acetylation, methylation, glycosylation, phosphorylatio n,sumoylation, amidation, lipidation, and/or methylation. In various embodiments, a mini-nucleosom coree protein can be pegylated. id="p-203" id="p-203" id="p-203" id="p-203" id="p-203"

[0203] In some embodiments, a. mini-nucleosome core protein is modified by association of the mini-nucleosome core protein with polyethylene glycol (PEG). PEG are nonionic, nontoxic, biocompatible and. highly hydrophilic polymers. PEG is mostly used, for the covalent modification of biological macromolecules and surfaces. PEG conjugation increases the apparent, size of the polypeptide, thus reducing the renal filtrati andon altering biodistribution. PEGylation of peptides can enhance therapeutic properties due to their increased solubilit (fory hydrophobic 85 peptides), prolonged half-life through reduce renad lclearance, and masked antigenici forty minimum immune response in the host. PEGs of varying PEG chain lengths have been used in FDA cleared drug withs molecular weights rangin gfrom 5-40 kDa. In Figure s1, 3, 4, 5 and 6, we show' schematics of how PEGs of varying PEG chain lengt canhs be utilized to provide mini- nucleosome core proteins of varying size. id="p-204" id="p-204" id="p-204" id="p-204" id="p-204"

[0204] Many current particles use PEG of size lOkDa or larger howeve, r, a drawback to using larger PEG size is that it also increases particle size. (Feuz L. et al. 2007). The present disclosure provides, among other things, particles with varying PEG length to formulate mini- nucleosomes with varying size- preferably small erthan 20nm in diameter In. Figure 1, we show a minima lPEG length of 12 chains and how it can be utilize tod modify amino acids in the mini- nucleosome core proteins. The final size of the loaded mini-nucleosome also depends on the PEG size used to modify the mini-nucleosom coree proteins. Figur e2 shows that by attaching PEG12, the molecul weightar of the peptide increases accordingl y,however doesn’t change the physical characteristics such as solubilit ofy the peptide. id="p-293" id="p-293" id="p-293" id="p-293" id="p-293"

[0293] In some embodiments, a mini-nucleosome core protein can have a total molecular weight between 1700g/mol and 20000 g/mol, e.g., 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, or 20000 g/mol. In various embodiments, a mini-nucleosome core protein can have a total molecul weightar between lOOKda and 10,000 kDa, e.g, 100, 200, 500, 1000, 2,000, 3000, 5000, 8000, and 10000 kDa. id="p-206" id="p-206" id="p-206" id="p-206" id="p-206"

[0206] The amino acid sequence may be used in reverse or in any order .One may also contemplate changing one or non-essential amino acid in the domain to obtain same charge or other properties of the domain. For the avoidance of doubt, any mini-nucleosome core protein provided herein, including the exemplar7 ymini-nucleosome core proteins provided in Table 13 below, can be modified at any amino acid and/or with any of one or more of the modificatio ns provided herein.

Table 13.

Exemplary Mini-nucleosom coree protei nsequences SEQ Net Number Molecular Iso-electrie ID charge of weigh t point (pH) NO. at pH7 residues (g/mo0 86 KRHRKLREKRHRKLRRRRRLKRHRKKRHRKLREK 336 22.4 34 4773.77 12.72 KRHRKGSSLREKRHRKLRRRRRLKRHRKKRHRKLR 337 EGGSK 22.4 40 5206.16 12.72 KRHRKREGS SLREKRHRKNDLRRRRRLKRHRKKRH 338 RKLREGGSK 21.4 44 5720.65 12.46 KKPKKREGS SLREKRHRKNDLRRRRRLKRHRKKRH 339 RKLREGGSK 21.3 44 5624.6 12.38 RRLARRGSSLREKRHRKLRRRRRLKKPKKKRHRKLR 340 EGGSK 22.2 41 5213.23 12.72 KRHRKLREKRHRKLREKRHRKLKRHRKKRHRKLRE 341 K 21.5 36 4984 12.48 KRHRKRILREKRHRKLREARKRHRKLKRHRKKRHR 342 KLREK 23.5 40 5480.61 12.56 KRHRKKGKKKKGEKGKKKLKGKKKLRRRRRRRQR 343 R 25.1 35 4507.55 12.78 KRHRKAPAPKGKKKKGEKGKKKLKGKKKLKPKPRR 344 RRRRRQRR 27.1 43 5294.51 12.79 KRHRKGGSGGKGKKKKGEKGKKKLKGKKKLARRR 345 RRRRQRR 25.1 41 4893.91 12.78 KRHRKLREKRHRKRRRRRRRKRHRKLREKRRQRR 346 24.3 34 4906.85 12.84 KRHRKKRHRKKRVKKKRHRKRRRRRRD SLL 347 21.3 30 4141.02 12.86 KRHRKKRHRKYQKRVKKKRHRKS S SRRRRRRD SLL 348 21.3 35 4693.55 12.64 349 KRHRKKKEEEKKEEEKKEEEKRRRRRRRQRRR 12.1 32 4473.09 11.61 KRHRKWRKKEEEKKEEEKKEEEKRIRRRRRRRQRRR 350 14.1 36 5084.83 11.79 KRHRKRGDKRHRKRRRRRKRHRKTPHKKK 351 20.4 29 3964.72 12.82 KRHRKFIRGDKRHRKRRRRRKRHRKLATPHKKK 352 20.4 33 4409.28 12.82 KRHRKRGDKRHRKRRRRRKRHRKGS SRNTPHQKKK 353 K 22.4 36 4722.51 12.86 KRHRKRGDKRHRKLKRHRKRRRRKRHRKTPHKK 354 22.5 33 4499.37 12.86 KRHRKRGDKRHRKKRHRKKRHRKRGDKKTK 355 19.4 30 3983.71 12.5 I KRHRKRKRKRKRRRRRKKKRASSLNIAKRRRR 357 24.1 32 4308.23 13.26 KRKKRKGKRLKRRREKRHRKRASSLNIAKKKK 358 20.1 32 4054.95 12.68 KRKKRRLKRKRKRRRRREKRHRKRRRQRRRKK 359 27.1 32 4618.63 13.01 KRKKRRKRKRRRRRKRHRKLRERKRRLREKK 360 24.1 31 4420.4 12.75 87 I KRHRKWRHRARSKRHRKKKKKKRKKRKGK 362 22.3 29 3902.77 13.03 KRHRKRGDKRHRKKKKNRRKKRALRKKRKGK 363 22.2 31 4047.92 12.73 KKRKRGGKTKKKAKKALKKKKKGKKKKRRRRKKA 364 APKK 28 38 4541.77 12.87 KKKAYPKALKKPKKKKKAYPKALKRRRRRKNRRK 365 KRALKRHRK 29.1 43 5481.83 12.53 KTRSKKKKKRGDKKKKNRRKKRALNTQIHKKKKKA 366 APKK 23.1 39 4725.78 12.4 KGKKKKGEKGKKKLKGKKKLRRRRRSPKKRRQRR 367 23 34 4242.23 12.68 KRHRKLREKRHRKLRRRRRLKRHRKKRHRKLREK 368 22.4 34 4773.77 12.72 KRHRKLREKRHRKLREKRHRKLKRHRKKRHRKLRE 369 K 21.5 36 4984 12.48 KRHRKKGKKKKGEKGKKKLKGKKKLRRRRRRRQR 370 R 25.1 35 4507.55 12.78 KRHRKLREKRHRKRRRRRRRKRHRKLREKRRQRR 371 24.3 34 4906.85 12.84 KRHRKKRHRKKRVKKKRHRKRRRRRRD SEE ר 72 21.3 30 4141.02 12.86 KRHRKKKEEEKKEEEKKEEEKRRRRRRRQRRR 373 12.1 32 4473.09 11.61 KRHRKQSKKEEEKKEEEKKEEEKNQRRRRRRRQRR 374 R 12.1 36 4930.53 11.61 KRHRKRGDKRHRKRRRRRKRHRKTPHKKK 375 20.4 29 3964.72 12.82 KRHRKRGDKRHRKLKRHRKRRRRKRHRKTPHKK 376 22.5 33 4499.37 12.86 3-י ר KRHRKRGDKRHRKKRHRKKRHRKRGDKKTK 19.4 30 3983.71 12.5 KRHRKRGDKKRKKKKRGDKKRRRRRKKKPPSY 378 21.1 32 4172.01 12.33 KRHRKGGSRGDKKRKKKKRGDSSSKKRRRRRKKKP 379 PSY 21.1 38 4634.43 12.33 KRHRKRKRKRKRRRRRKKKR AS SLNIAKRRRR 380 24.1 32 4308.23 13.26 KRKKRKGKRLKRRREKRHRKRASSLNIAKKKK 381 20.1 32 4054.95 12.68 KRKKRRLKRKRKRRRRREKRHRKRRRQRRRKK 382 27.1 32 4618.63 13.01 KRKKRRKRKRRRRRKRHRKLRERKRRLREKK 383 31 4420.4 pH 12.75 24.1 KRKNGRKRKRKKRHRKKKKRRRRKRHRKNGRKKK 384 28.2 34 4587.61 13.2 385 KRKWRNGRKRKRQKRHRKKKKRARRRRKRHRKNG RKHKKK 30.3 40 5422.54 13.26 KRHRKWRHRARSKRHRKKKPKKRKKRKGK 386 21.3 29 3871.71 13.03 88 KRHRKPKPRIWRHRARSRDKRHRKKKPKKRKKRKG 387 23.3 36 4734.73 12.78 K 11.77 KKKRKLRGDLKRKGSSYQPLAPAPKKKRKRGDKRK 388 16.0 41 4894.87 LFYQPL id="p-207" id="p-207" id="p-207" id="p-207" id="p-207"

[0207] For the avoidance of doubt, the present disclosure includes modified mini - nucleosome core proteins including, withou limitatt ion a mini-nucleosome core protein of the present disclosure in which at least one amino acid of the mini-nucleosom coree protein includ es a modification disclosed herein.

Modifie dMim-Nudeosome Core Proteins id="p-208" id="p-208" id="p-208" id="p-208" id="p-208"

[0208] The present disclosure provides, among other things, mini-nucleosome core proteins that include at least one modified amino acid residue. Various modifications and certain advantageou thereofs are provided throughout the present disclosure. For the avoidance of doubt, the present disclosure provides that any and/or all residues of any and/or all domains, mini-nucleosom coree proteins or, other polypeptides provide dherein, and/or any and all residues of any portion(s) thereof, can be modified in accordance with the present disclosure of modifications. Moreover ,various advantages resulting from any such amino acid modification as disclosed herein do not depend upon the specific residu ewithin a domain, mini-nucleosome core protein, or other polypeptide that is modifie d,and/or the position thereof within a domain, mini-nucleosom coree protein, or other polypeptid e.In various embodiments various, advantages resulting from any such amino acid modification as disclosed herein are realized upon inclusion of the modification at any of one or more residues of a domain, mini-nucleoso me core protein, or other polypeptide in accordance with the present disclosure. id="p-209" id="p-209" id="p-209" id="p-209" id="p-209"

[0209] In various embodiments, a modification of an amino acid present in a domain, mini-nucleosome core protein, or other polypeptide can be any of: (i) phosphorylation; (ii) sulfation; (iii) glycosylation (e.g., N-glycosylation, C-glycosylation, and/or O-glycosylation); (iv) prenylation (e;g., geranylatio and/orn farnesylation); (v) methylation; 89 (vi) sialylation; (vii) lipidation and/or lipoylation; (viii) acetylation; (ix) hydroxylation; (x) palmitoylation; (xi) mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylatio and/orn; (xv) any combination thereof, including any numbe ofr one or more of the modifications or variants thereof, e.g., in a branched or unbranched modification chain. id="p-210" id="p-210" id="p-210" id="p-210" id="p-210"

[0210] Examples of modifications and modified mini-nucleosom coree proteins are found, e .g., in Figures 20-31. Those of skill in the art will appreciate that even, absent express disclosur ofe the atoms of any given chemical linkage between a modification and amino acid, or between two modifications in a modification chain, that such linkages are well known in the art of amino acids and amino acid modifications.

Phosphorylated Mini-Nucleosome Core Proteins id="p-211" id="p-211" id="p-211" id="p-211" id="p-211"

[0211] Phosphorylatio typicallyn occurs on serine, threonine, tyrosine and/or, histidine residues (S, Y, T, and/or H; see, e.g., Fig. 27). Phosphorylatio nincludes covalent linkage of a phosphate group to an amino acid residue, such as to a hydroxyl side chain of a serine residue, a hydroxyl side chain of a threonine residue, or a phenolic side chain of a tyrosine residue.

Phosphorylatio nis known to mediate protein functions including target binding, cellular localization, and enzymat icactivity, among others. The present disclosure includes modified mini-nucleosom coree proteins in which one or more threonine, tyrosine, and/or histidine residues are phosphorylate d,e.g., mono-phosphorylated or bis-phosphorylated. In some embodiments, a modified mini-nucleosom coree protein is phosphorylated by incorporation of protected phosphor-amino acids during polypeptide synthesis. In some embodiments, a modified mini-nucleosom coree protein is phosphorylated by post-synthesis phosphorylation of residues, 90 e.g., serine, threonine, or tyrosine residues. In general, at least certain advantages of phosphorylation have been demonstrated, e.g., by Kobayashi et. Al. 1996 (showing phosphorylation of ATF-1 increased DNA bindin gcapabilities), Robin et. Al, 2003 (showing phosphorylation of GSTA4-4 increased targeting of GSTA4-4 to mitochondri bya interaction with mitochondrial surface proteins), Rossetto D et al. 2012 (showing that phosphorylation of extra-nucleosomal histone Hl, a linker Histone increases, stabilization of the nucleosome), and Anai et al, 2007 (showing a bis-phosphorylate peptided with increased binding affinit andy selectivity for WW domains). id="p-212" id="p-212" id="p-212" id="p-212" id="p-212"

[0212] In various embodiments, phosphorylation of one more residues of a modified mini-nucleosom coree protein increases the stability of the modified mini-nucleosome core protein, or loaded mini-nucleosom coree proteins including the same, as compared to a reference mini-nucleosom coree protein that does not include one or more of the phosphorylation modifications. In various embodiments phosphorylation, of one more residues of a modified mini-nucleosom coree protein increases the half-life and/or bioavailabili ofty the modified mini - nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the phosphorylation modifications. In various embodiments, phosphorylation of one more residues of a modified mini-nucleosom coree protein increases the affinity or avidity with a target cell or other bindin gpartner of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the phosphorylation modifications. In various embodiments, phosphorylation of one more residues of a modified mini-nucleosom coree protein increases the rate at which the modified mini-nucleosome core protein, or loaded mini-nucleosom coree proteins including the same, enters one or more target cells (i.e., crosses the cell membranes of target cells) as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the phosphorylation modifications. In various embodiments, phosphorylation of one more residues of a modified mini-nucleosome core protein increases the affinity or avidity of the mini-nucleosom coree protein with one or more nucleic acid cargos as compared to a reference mini-nucleosom coree protein that does not include one or more of the phosphorylation modifications. Thus, in some embodiments, phosphorylation of one more residues of a modified 91 mini-nucleosom coree protein increases deliver ofy a mini-nucleosome core protein, loaded mini-nucleosom coree protein, or nucleic acid cargo of a mini-nucleosom coree protein to a target cell, tissue, or organ. id="p-213" id="p-213" id="p-213" id="p-213" id="p-213"

[0213] In certain particular embodiments of the present disclosur e,phosphorylation of one more residues of a modified mini-nucleosom coree protein decreases accumulation in liver of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a reference mini-nucleosome core protein that does not include one or more of the phosphorylation modifications. This is particularly advantageou in s view of the empirical observation that most molecules and drug products, including adeno- associated viruses injected intravenously, accumulate in liver cells. Thus, phosphorylated mini - nucleosome core proteins of the present disclosure advantageously provide a means of nucleic acid deliver thaty reduces accumulation in liver of mini-nucleosome core proteins and/or loaded mini-nucleosome core proteins. id="p-214" id="p-214" id="p-214" id="p-214" id="p-214"

[0214] In certain particular embodiments of the present disclosur e,targeting of a phosphorylated mini-nucleosome core protein, or loaded mini-nucleosome core protein including the same, to a target, target cell, or target tissue, e.g., by inclusion in the mini-nucleosom coree protein of a targeti ngdomain as provide dherein, is increased in affinit avidiy, ty, or rate as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the phosphorylation modifications. For example, phosphorylated mini-nucleosome core proteins, and loaded mini-nucleosome core proteins including the same, that are targeted to neurons, e.g., by inclusion of targeting domain for neuronal targeting, can deliver nucleic acid cargos to neurons with increased affinit avidiy, ty, or rate as compared to a referenc mini-nucleosomee core protein that does not include one or more of the phosphorylation modifications. id="p-215" id="p-215" id="p-215" id="p-215" id="p-215"

[0215] Exemplary domains that can be included in mini-nucleosom coree proteins and, which can optional lybe modified by phosphorylation at one more residues, are provide din below Tables 14-16. id="p-216" id="p-216" id="p-216" id="p-216" id="p-216"

[0216] Table 14 includes exemplary targeting domains that target neurons and, which target neurons with greater affinit avidiy, ty, or rate when phosphorylated at one or more underline serine,d threonine, and/or tyrosine residues. 92 id="p-217" id="p-217" id="p-217" id="p-217" id="p-217"

[0217] Table 15 includes exemplary targeting domains that target muscle cell s,and which target muscle cells with greater affinit avidity, y,or rate when phosphorylated at one or more underline seridne, threonine, and/or tyrosine residues. id="p-218" id="p-218" id="p-218" id="p-218" id="p-218"

[0218] Table 16 includes exemplary targeting domains that target endothelial cells, and which target endotheli cellsal with greater affinit avidity, y,or rate when phosphorylated at one or more underline seridne, threonine, and/or tyrosine residues.

Table 14 Exemplary mini-nucleosome core protein domain SEQID NO.

KKRHRKYPKKSRRSRLRNFRGDYNQYTRRRRR 397 KRKKRHRKRIRGRDVKYSYARKRHRKFQKWNYK 398 KKRHRKARRVTALREGRRHRKGERRRRRPPSY 399 KKRHRKALGSSDSLLARKRHRKKRKRKKRHRK 400 KKRHRKGSSKKRPKPRKKRHRKKRHRKKRHRKLL 401 KKRHRKRIQRRSRRGSSKHKGRDVILKKDVRKRHRK 402 KKRHRKKKDGKKRKRLLRKKHARALYIGSRKRGRKP 403 KKRHRKPPKDGEAQPKRHRKRRRRRKRHRKLRA 404 KKRKKRHRKLARGPRVARKRHRKRRRRRDRYQRL 405 Table 15 Exemplary mini-nudeosome core protein domain SEQ ID NO.

KKRHRKRGFRRVSRRRGKKKEQRRERNARGKKGKRHRK 406 KKRHRKRRQPPRSISSHPLRKKRKGKTRRLRGDLRNSRR 407 KKRHRKRLRKKRKGKGSRPGSGFVKKTKQRRRRR 408 KKRHRKHRTKSGRSRIRKKRKGKRHARKKRRQRRRPPSY 409 KKRHRKKPVNRWSARNRRKKRALLRRRHYQRL 410 KKRHRKRKYKQCHKKGGHCFPKEKARRKKRKGKNEI 411 KKRHRKRIKKYRYYLKPLKKKRKKRKGKRHYLIIR 412 KKRHRKDRGRKKRRQRRRPQKPRKKRRQRRFQQI 413 93 KKRHRKGSSDPPRDOPFHRKRHRKKRHRKKRHRGRR 414 KKRHRKARSKTIWHPQSTPYKRHRKRKK^ 415 RKKRKGKRAKRHRKKRHRKKPKNMTPYRSPPPYVPP 416 Table 16 Exemplary mini-nudeosome core protein domain SEQ ID NO.

KKKRKRGDKRKRKRHRKKKRRRRLSIPPKA 417 KKKRKRGDKRKRKRHRKKKRRRRFQTPPQL 418 KKKRKRGDKRKRKRHRKKKRRRRLTPATAI 419 KKKRKRGDKRKRKRHRKKKRRRRSIGYPLP 420 KKKRKRGDKRKRKRHRKKKRRRRCLIRRTSIC 421 KKKRKRGDKRKRKRHRKKKRRRRCFFWKFRWMC 422 Sulfated Mini-Nucleosome Core Proteins id="p-219" id="p-219" id="p-219" id="p-219" id="p-219"

[0219] Sulfation refers to the covalent linkage of sulfate to a tyrosine (Y) residue (see, e.g., Fig. 25). In certain embodiments, an amino acid immediately N-terminal to a sulfated tyrosine in a polypeptide is an amino acid selected from E, N, S, H, V, and D, and/or an amino acid immediately C-terminal to a sulfate tyrd osine in a polypeptide is an amino acid selected from E, L, D, Q, P, T, R and Y. In general, sulfati onis known to increas ethe affinity and/or avidity of protein-protei intern actions. For example, sulfati onis predicted to increase select in bindin forg increased update into endothelial cells, and Farzan et al. 1999 demonstrated that sulfati onof CCR5 facilitates HIV entry. Proteins known to undergo sulfati oninclude G-protein- coupled receptors, adhesion molecules, hormones, and extracellular matrix proteins .Sulfation is known to contribute to L- and P-selectin-mediated neutrophil recruitment, and leukocyte rolling (Somers et al, 2003). id="p-220" id="p-220" id="p-220" id="p-220" id="p-220"

[0220] In various embodiments, sulfation of one more residues of a modified mini- nucleosome core protein increases affinity or avidity with a target cell or other binding partner of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the sulfation modifications. In various embodiments, sulfati onof one more residues of a 94 modified mini-nucleosom coree protein increases affinity or avidity of binding with a target receptor of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a reference mini-nucleosome core protein that does not include one or more of the sulfati onmodifications. Thus, in some embodiments, sulfati onof one more residues of a modified mini-nucleosome core protein increases delivery of a mini - nucleosome core protein, loaded mini-nucleosome core protein, or nuclei acidc cargo of a mini - nucleosome core protein to a target cell, tissue, or organ. id="p-221" id="p-221" id="p-221" id="p-221" id="p-221"

[0221] In various embodiments, sulfation of one more residues of a modified mini- nucleosome core protein increases the rate at which the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, enter ones or more target cells (i.e., crosses the cell membranes of target cells) as compared to a referenc mini-nucleosomee core protein that does not include one or more of the sulfation modifications. id="p-222" id="p-222" id="p-222" id="p-222" id="p-222"

[0222] In various embodiments, sulfation of one more residues of a modified mini- nucleosome core protein increases blood-brain barrier penetrat ionby the modified mini- nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the sulfati onmodifications. id="p-223" id="p-223" id="p-223" id="p-223" id="p-223"

[0223] In various embodiments, sulfation of one more residues of a modified mini- nucleosome core protein increases selectivity of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, for target receptors and/or, selectivity of internalization afte receptorr binding, as compared to a reference mini-nucleosom coree protein that does not include one or more of the sulfation modifications. id="p-224" id="p-224" id="p-224" id="p-224" id="p-224"

[0224] Table 17 includes exemplary sulfated domains for inclusion in mini-nucleoso me core proteins that enhanc ebindin tog endothelial cell surfac emarker sand/or increase update of mini-nucleosom coree proteins and/or loaded mini-nucleosome core proteins by endotheli al cells.

Table 17 Exemplary smm-mideesome core protein domain SEQ IB NO.

KKKRKRGDKRKRKRHRKKKRRRREYYLSIPPKA 423 KKKRKRGDKRKRKRHRKKKRRRDYRFQTPPQL 424 95 KKKRKRGDKRKRKRHRKKKRRRHYRLTPATAI 425 KKKRKRGDKRKRKRHRKKKRRRRVYQSIGYPLP 426 KKKRKSYRRGDKRKRKRHRKKKRRRRCLIRRTSIC 427 KKKRKEYRGDKRKRKRHRKKKRRRRCFFWKFRWMC 428 Glycosylated Mini-Nucleosome Core Proteins id="p-225" id="p-225" id="p-225" id="p-225" id="p-225"

[0225] Glycosylation includes, among other things, N-glycosylation, O-glycosylation, and C-glycosylati on.N glycosylation, refers to covalent linkage of a glycan to a nitrogen atom of a side chain of an amino acid, typicall they amide nitrogen of an asparagine (N) residue.

Examples of N glycosylati oninclude GlcNAc־P־Asn, GlcNac-a-Asn, and Glc-Asn. O- glycosylation refers to covalent linkage of a glycan to an oxygen atom of a hydroxyl side chain of an amino acid, typically an oxygen atom of a hydroxyl side chain of a serine (S) or threonine (T) residue. Examples of O-glycosylation include GlcNac־P־Ser/Thr and GalNac-a-Ser/Thr. C- glycosylation refers to covalent linkage of mannose to a carbon atom of a side chain of an amino acid, typicall ay carbon atom of a tryptophan (W) residue. An example of C-glycosylation is a- mannosyl tryptopha n.Accordingl y,in various embodiments glycosylation, can refer to one or more modifications, each of which modifications can be any of N-glycosylation, C- glycosylation, or O-glycosylation. Known functions of glycosylation modifications include contributions to protein folding and cell signaling. In general, glycosylation is also known to improve protein stability and serve as an epitope for association with binding partners. id="p-226" id="p-226" id="p-226" id="p-226" id="p-226"

[0226] In various embodiments, glycosylation of one more residues of a modified mini- nucleosome core protein increases the stability of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosome core protein that does not include one or more of the glycosylation modifications. In some embodiments, glycosylati increaseson stability of the modified mini-nucleosome core protein, or loaded mini-nucleosom coree proteins including the same, in that physical properties of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, including protein structur ande protein charge, are maintained and/or maintained for a longer period of time, e.g., afte administrationr to a subject. In some embodiments, glycosylation decreases the occurrenc eor rate of thermal and/or kineti c denaturatio ofn the modified mini-nucleosome core protein, or loaded mini-nucleosom coree 96 proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the glycosylation modifications. id="p-227" id="p-227" id="p-227" id="p-227" id="p-227"

[0227] In various embodiments, glycosylation of one more residues of a modified mini- nucleosome core protein increases half-life and/or bioavailabilit ofy the modified mini - nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the glycosylation modifications. id="p-228" id="p-228" id="p-228" id="p-228" id="p-228"

[0228] In various embodiments, glycosylation of one more residues of a modified mini- nucleosome core protein increases affinity or avidity with a target cell or other binding partner of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the glycosylation modifications. In various embodiments, glycosylation of one more residues of a modified mini-nucleosom coree protein increases affinity or avidity of bindin withg a target receptor of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the glycosylation modifications. Thus, in some embodiments, glycosylation of one more residues of a modified mini-nucleosome core protein increases delivery of a mini-nucleosome core protein, loaded mini-nucleosome core protein, or nucleic acid cargo of a mini-nucleosom coree protein to a target cell, tissue, or organ. id="p-229" id="p-229" id="p-229" id="p-229" id="p-229"

[0229] In various embodiments, glycosylation of one more residues of a modified mini- nucleosome core protein increases the rate at which the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, enter ones or more target cells (i.e., crosses the cell membranes of target cells) as compared to a referenc mini-nucleosomee core protein that does not include one or more of the glycosylation modifications. id="p-230" id="p-230" id="p-230" id="p-230" id="p-230"

[0230] In various embodiments, glycosylation of one more residues of a modified mini- nucleosome core protein decreases precipitation and/or aggregation of the modified mini- nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the glycosylation modifications. id="p-231" id="p-231" id="p-231" id="p-231" id="p-231"

[0231] Table 18 includes exemplary glycosylated domains, in particular galactose- modified targeti ngdomains that target mini-nucleosom coree proteins to liver.

Table 18 Exemplary mini-nudeosome core protein domain SEQ ID NO.

KKRHRKARARKKAAKARIKKAAPAKKAANRARKKH 429 KKRHRKGSSRRPRPGTGPGRRPRPRPRPRKKRNRSRQRRR 430 KKRHRKKYKQKIKHVVKLKKHRKRKRNRSIKVAV 431 KKRHRKSSSRTLQAHHDRQSNKRKRKNRSRRRRR 432 KKRHRKRNRSIHFNPRHRRRRRRDVARARAEKSKKK 433 KKRHRKNRSKKQRFRHRNRKGYRSQRGHSRGRNQNSRR 434 PrenylatedMini-Nucleosome Core Proteins id="p-232" id="p-232" id="p-232" id="p-232" id="p-232"

[0232] Prenylati ontypicall occury s on cysteine residues (see, e.g., Fig. 26 for an example of a prenylated mini-nucleosome core protein). Prenylati onincludes covalent linkage of a lipid chain famesyl (CIS) or geranylgera nyl(C20) isoprenoid moiet yto a free thiol group of a cysteine residue. Accordingl y,prenylation includes modifications including farnesylation, geranylation, and geranylgeranylation. Prenylati onis often found on C-termina resl idues of polypeptide ins; mammals, approximately 2% of proteins are prenylated at thei rC-terminal residues. Prenylati onof a polypeptide such as a mini-nucleosome core protein significantl y impacts hydrophobicity of the polypeptide. For at least that reason, prenylation of a mini - nucleosome core protein increases the strengt (e.g.,h affinity or avidity) of interactio betweenn a mini-nucleosom coree protein and plasma membranes, facilitatin efficientg cellular uptake .

Prenylati onof a polypeptide such as a mini-nucleosome core protein also increases cell penetrat ingability and/or uptake by target cells (see, e.g., Ochocki ID et al, 2011). Prenylati on has also been shown to strengthen (e.g., increase the affinit ory avidity of) protein-protei n interactions (e.g., promoting protein-protein and protein-membrane interactions of proteins such as Ras, Rho, and Rab, etc., as reported by Gelb et al, 1998). Prenylati onof proteins is also known to facilita tehoming to target subcellular localizations. id="p-233" id="p-233" id="p-233" id="p-233" id="p-233"

[0233] In various embodiments, prenylation of one more residues of a modified mini - nucleosome core protein increases affinity or avidity with a target cell or other binding partner of 98 the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the prenylation modifications. In various embodiments, prenylation of one more residues of a modified mini-nucleosom coree protein increases affinity or avidity of bindin withg a target receptor of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the prenylation modifications. Thus, in some embodiments, prenylation of one more residues of a modified mini-nucleosome core protein increases delivery of a mini-nucleosom coree protein, loaded mini-nucleosome core protein, or nucleic acid cargo of a mini-nucleosom coree protein to a target cell, tissue, or organ. id="p-234" id="p-234" id="p-234" id="p-234" id="p-234"

[0234] In various embodiments, prenylation of one more residues of a modified mini - nucleosome core protein increases strength (e.g., affinit ory avidity) of association with cell membranes of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the prenylation modifications. In various embodiments, prenylation of one more residues of a modified mini-nucleosom coree protein increases the rate at which the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, penetrates cell membranes as compared to a referenc mini-nucleosomee core protein that does not include one or more of the prenylation modifications. In various embodiments, prenylation of one more residues of a modified mini-nucleosome core protein increases the rate at which the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, enters one or more target cells (i.e., crosses the cell membranes of target cells) as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the prenylation modifications. id="p-235" id="p-235" id="p-235" id="p-235" id="p-235"

[0235] In various embodiments, prenylation of one more residues of a modified mini - nucleosome core protein increases the rate at which the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, home to target subcellular localization as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the prenylation modifications. 99 id="p-236" id="p-236" id="p-236" id="p-236" id="p-236"

[0236] Table 19 includes exemplary prenylated domains, in particula prenylar ted targeting domains that target mini-nucleosome core proteins to liver cells and other cell types.

Table 19 Exemplary mini-nudeosome core protein domain SEQ ID NO.

KKRHRKARARKKAAKARIKKAAPAKKAARACIILRKKH 435 KKRHRKGSSRRPRPGTGPGRRPRPRPRPRKKRCASERQRRR 436 KKRHRKCIIEKYKQKIKHVVKLKKHRKRKRIKVAV 437 KKRHRKCQALSSSRTLQAHHDRQSNKRKRKRRRRR 438 KKRHRKRIHFNPRHRRRRRRCIAEDVARARAEKSKKK 439 KRHRKKKQRFRHRNRKGYRSQRGHSRGRNQNSRRCIILR 440 Methylated Mini-Nucleosome Core Proteins id="p-237" id="p-237" id="p-237" id="p-237" id="p-237"

[0237] Methylation typically occurs on lysine (K) and arginine (R) residues (see, e.g., Figure 28). Methylated residues include mono-methylate residues,d di-methylate residd ues, and tri-methylated residue s,among others. In particula lysiner, residues are typically mono- methylated, di-methylate ord, tri-methylated, and arginine residues are typicall mono-y methylated or di-methylated. Studie haves demonstrated that histones methylated on certain residues cause and/or contribute to epigenetic contro lof gene expression. Methylation of polypeptides such as methylated mini-nucleosome core proteins increases targeting of the polypeptides to the nucleus. id="p-238" id="p-238" id="p-238" id="p-238" id="p-238"

[0238] In various embodiments, methylation of one more residues of a modified mini- nucleosome core protein increases the rate of delivery to the nuclei of target cells of the modified mini-nucleosom coree protein, or loaded mini-nucleosom coree proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the methylat ionmodifications. In various embodiments, methylation of one more residues of a modified mini-nucleosom coree protein increases the rate of deliver toy the nuclei of target cells of a nucleic acid cargo of a loaded mini-nucleosom coree protein including the modified mini- nucleosome core protein, as compared to deliver ofy the nucleic acid cargo of a referenc loadede mini-nucleosom coree protein that includes a mini-nucleosom coree protein that does not include one or more of the methylation modifications. In various embodiments, methylat ionof one more 100 residues of a modified mini-nucleosom coree protein increases expression in target cells of a coding sequenc ofe a nuclei acidc cargo of a loaded mini-nucleosome core protein including the modified mini-nucleosom coree protein, as compared to expression of a coding sequenc ofe a nucleic acid cargo of a referenc loadede mini-nucleosome core protein that includes a mini- nucleosome core protein that does not include one or more of the methylation modifications. id="p-239" id="p-239" id="p-239" id="p-239" id="p-239"

[0239] Table 20 includes exemplary methylated domains, in particular methylated targeting domains that target mini-nucleosome core proteins to cell nuclei and/or increase expression of nucleic acid cargos when included in a mini-nucleosome core protein of a loaded mini-nucleosome core protein.

Table 20 Exempiary mini-micleosome core protein domain SEQ11) NO.

KKRHRKARARKKAAKARIKKAAPAKKAARARKKH 441 KKRHRKGS SRRPRPGTGPGRRPRPRPRPRKKRRQRRR 442 KKRHRKKYKQKIKHVVKLKKHRKRKRIKVAV 443 KKRHRKSSSRTLQAHHDRQSNKRKRKRRRRR 444 KKRHRKRIHFNPRHRRRRRRDVARARAEKSKKK 445 KRHRKKKQRFRHRNRKGYRSQRGHSRGRNQNSRR 446 Sialylated Mini-Nucleosome Core Proteins id="p-240" id="p-240" id="p-240" id="p-240" id="p-240"

[0240] Sialylation refers to the covalent addition of sialic acid to the terminal end of a glycoprote oligosaccharidein chain. Sialylatio cann occur, e.g., on asparagine (N) and serin e(S) residues. Sialylatio cann increase endothelial cell targeti ngand/or blood brain barrier penetration. In some instance endothes, lial cell targeting provides a means of increased blood brain barrier penetration. id="p-241" id="p-241" id="p-241" id="p-241" id="p-241"

[0241] In various embodiments, sialylation of one more residues of a modified mini- nucleosome core protein increases blood-brain barrier penetrat ionby the modified mini- nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the sialylation modifications. 101 id="p-242" id="p-242" id="p-242" id="p-242" id="p-242"

[0242] In various embodiments, sialylation of one more residues of a modified mini- nucleosome core protein increases strength (e.g., affinit ory avidity) of association with cell membranes (e.g., endothelial cell membranes) of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosome core protein that does not include one or more of the sialylation modifications. In various embodiments, sialylation of one more residues of a modified mini-nucleosom coree protein increases the rate at which the modified mini-nucleosome core protein, or loaded mini - nucleosome core proteins including the same, penetrates cell membranes (e.g., endothelial cell membranes) as compared to a reference mini-nucleosom coree protein that does not include one or more of the sialylation modifications. In various embodiments, sialylation of one more residues of a modified mini-nucleosom coree protein increases the rate at which the modified mini-nucleosom coree protein, or loaded mini-nucleosom coree proteins including the same, enter ones or more target cells (i.e., crosses the cell membranes of target cells), such as endothelial cell s,as compared to a referenc mini-nucleosomee core protein that does not include one or more of the sialylation modifications. id="p-243" id="p-243" id="p-243" id="p-243" id="p-243"

[0243] Table 21 includes exemplary sialylated domains, in particular sialylat edtargeting domains that increas eendothelial cell targeting and/or blood brain barrier penetrat ionwhen included in a mini-nucleosome core protein.

Table 21 Exemplary mini-nudeosome core protein domain SEQ ID NO.

KKRHRKGGSLLRGEKELKRPPRRRRRKYIGSR 447 KKKRKLRGDLKRKPLISRRLIDRYQKKKRKRGDKRK 448 KKKRKLRGDLKRKSSSVRKKPGGSKKKRKRGDKRK 449 KKKRKLRGDLKRKGTQPEHSSTDHKKKRKRGDKRK 450 KKKRKRGDKRKRKRHRKKKRRRRLSIPPKA 451 KKKRKRGDKRKRKRHRKKKRRRRFQTPPQL 452 KKKRKRGDKRKRKRHRKKKRRRRNRSLTPATAI 453 KKKRKRGDKRKRKRHRKKKRRRRSIGYPLP 454 455 KKKRKRGDKRKRKRHRKKKRRRRNRSCLIRRTSIC KKKRKRGDKRKRKRHRKKKRRRRNRSCFFWKFRWMC 456 102 KKKRKNRSRGDKRKRKRHRKKKRRRRIELLQARGC 457 KKKRKRGDKRKRKRHRKKKNRSRRRRREDV 458 459 KKKRKRGDKRKRKRHRKKKRRRRVHPKQHRGGSKGC Lipidated and/or Lipoylated Mini-Nucleosome Core Proteins id="p-244" id="p-244" id="p-244" id="p-244" id="p-244"

[0244] Amino acids can be covalently modified by linkage to a variety of lipids, including fatt acids,y isoprenoids, and cholesterol (see, e.g., Fig. 30). Fatty acids that can be linked to amino acid residues for lipidation and/or lipoylation include caprylic acid (C8), capric acid (CIO), lauric acid (C12), myristic acid (C14), palmitic acid (C16) or Stearic acid (C18).

Fatty acids can be conjugated to the N-terminus of a polypeptide or to a side-chain of a lysine residue. Cysteine residues can also be covalently linked to fatt acids.y Lipoylation is a form of acylation that includes linkage of an amino acid to a lipoate (C8) functional group. Lipoylation can occur, e.g., on lysine residues. In some instances, a polypeptide such as a mini-nucleosome core protein can be covalently linked to cholesterol. Cholesterol can be conjugated to a mini- nucleosome core protein at a N-terminal residu eor at a C-terminal residue, and/or at a cysteine residue. id="p-245" id="p-245" id="p-245" id="p-245" id="p-245"

[0245] Lipidation and/or lipoylati oncan contribute to polypeptide localization and function. Lipidation and/or lipoylation can improve pharmacokinetic properties of a polypeptide such as a mini-nucleosom coree protein, e.g., to increas ehalf-life in circulation. Lipidation and/or lipoylation, e.g., with long-chain fatt acids,y can also increase targeting of polypeptides such as a mini-nucleosome core protein (see, e.g., Hossieni et al., 2019, demonstrating that palmitoylation can significantly improve targeting in Wnt trafficking). Linkag eof a mini - nucleosome core protein to cholesterol can increase membrane anchoring and thereby increase delivery to target cells. id="p-246" id="p-246" id="p-246" id="p-246" id="p-246"

[0246] In various embodiments, lipidation and/or lipoylation of one more residues of a modified mini-nucleosome core protein increases affinity or avidity with a target cell or other bindin partnerg of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the lipidation and/or lipoylation modifications. In various embodiments, lipidation and/or lipoylation of one more residues of a modified mini-nucleoso me core protein increases affinity or avidity of binding with a target receptor of the modified mini 103 nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the lipidation and/or lipoylation modifications. Thus, in some embodiments, lipidation and/or lipoylation of one more residues of a modified mini-nucleosome core protein increases delivery of a mini-nucleosom coree protein, loaded mini-nucleosome core protein, or nucleic acid cargo of a mini-nucleosom coree protein to a target cell, tissue, or organ. id="p-247" id="p-247" id="p-247" id="p-247" id="p-247"

[0247] In various embodiments, lipidation and/or lipoylation of one more residues of a modified mini-nucleosome core protein increases half-life and/or bioavailabili ofty the modified mini-nucleosom coree protein, or loaded mini-nucleosom coree proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the lipidation and/or lipoylation modifications.

Acetylated Mini-Nucleosome Core Proteins id="p-248" id="p-248" id="p-248" id="p-248" id="p-248"

[0248] Acetylation can occur, e.g., on alanine (A), valine (V), and/or lysine (K) residues.

An acetyl group can be covalently linked to an a-amino group of the N-terminus of a polypeptid e or to an s-amino group of a lysine residue (see, e.g., Figs. 23 and 24). Acetylati onrefers to the substitution of hydrogen with an acetyl group in a residu eof a polypeptide, such as a mini- nucleosome core protein. Acetylation can increase the stability and/or biological activity of a polypeptide, such as a mini-nucleosom coree protein. For example, acetylation can increase stability of modified polypeptides by preventing N-terminal degradation. Acetylation is a reversible modification. id="p-249" id="p-249" id="p-249" id="p-249" id="p-249"

[0249] In various embodiments, acetylation of one more residues of a modified mini- nucleosome core protein increases the stability of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosome core protein that does not include one or more of the acetylation modifications. In some embodiments, acetylati onincreases stability of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, in that physical properties of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, including protein structur ande protein charge, are maintained and/or maintained for a longer period of time, e.g., afte administrationr to a subject .In some embodiments, acetylation 104 decreases the occurrence or rate of therma and/orl kinetic denaturatio ofn the modified mini - nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the acetylation modifications. id="p-250" id="p-250" id="p-250" id="p-250" id="p-250"

[0250] In various embodiments, acetylation of one more residues of a modified mini- nucleosome core protein increases half-life and/or bioavailabilit ofy the modified mini - nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the acetylation modifications.

Hydroxylated Mini-Nucleosome Core Proteins id="p-251" id="p-251" id="p-251" id="p-251" id="p-251"

[0251] Hydroxylation can occur, e.g., on proline (P) and lysine (K) residues, as well as other amino acids including without limitation asparagine aspartate, and histidine (see, e.g., Fig. 29). Proline and/or lysine hydroxylation is associated with a variety of physiological molecul es and/or processes, including certain molecules and/or process of certain pathological states. For example, proline hydroxylation can increase stability of a polypeptide such as a mini - nucleosome core protein. id="p-252" id="p-252" id="p-252" id="p-252" id="p-252"

[0252] In various embodiments, hydroxylation of one more residue s,e.g., one or more proline residue s,of a modified mini-nucleosom coree protein increases the stability of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-nuce leosome core protein that does not include one or more of the hydroxylation modifications. In some embodiments, hydroxylation of one more residue s,e.g., one or more proline residue s,increases stability of the modified mini-nucleoso me core protein, or loaded mini-nucleosome core proteins including the same, in that physical properties of the modified mini-nucleosom coree protein, or loaded mini-nucleosom coree proteins including the same, including protein structure and protein charge, are maintained and/or maintained for a longer period of time, e.g., afte administrationr to a subject. In some embodiments, hydroxylation of one more residues, e.g., one or more proline residue s,decrease s the occurrenc eor rate of therma and/orl kinetic denaturatio ofn the modified mini-nucleoso me core protein, or loaded mini-nucleosome core proteins including the same, as compared to a 105 referenc mini-nucleosomee core protein that does not include one or more of the hydroxylation modifications. id="p-253" id="p-253" id="p-253" id="p-253" id="p-253"

[0253] In various embodiments, hydroxylation of one more residue s,e.g., one or more proline residue s,of a modified mini-nucleosom coree protein increases half-life and/or bioavailabili ofty the modified mini-nucleosom coree protein, or loaded mini-nucleosom coree proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the hydroxylation modifications.

Palmitoylated Mini-Nucleosome Core Proteins id="p-254" id="p-254" id="p-254" id="p-254" id="p-254"

[0254] Palmitoylation is a lipid modification that typically occurs on cysteine I residues, and can also occur, e.g., on serin e(S) and threonine (T) residues. Palmitoylation includes the covalent attachment of a fatt acid,y such as palmitic acid, to a residu eof a polypeptide such as a mini-nucleosom coree protein. Palmitoylation can be presen t,e.g., on residues of membrane proteins. id="p-255" id="p-255" id="p-255" id="p-255" id="p-255"

[0255] In various embodiments, palmitoylation of one more residues of a modified mini - nucleosome core protein increases strength (e.g., affinit ory avidity) of association with cell membranes (e.g., endothelial cell membranes) of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosome core protein that does not include one or more of the palmitoylat ionmodifications.

In various embodiments, palmitoylation of one more residues of a modified mini-nucleoso me core protein increases the rate at which the modified mini-nucleosom coree protein, or loaded mini-nucleosom coree proteins including the same, penetrates cell membranes (e.g., endothelial cell membranes) as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the palmitoylat modification ions. In various embodiments palmitoylation, of one more residues of a modified mini-nucleosome core protein increases the rate at which the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, enters one or more target cells (i.e., crosses the cell membranes of target cells), such as endothelial cell s,as compared to a referenc mini-nucleosomee core protein that does not include one or more of the palmitoylation modifications.

Mannosylated Mini-Nucleosome Core Proteins 106 id="p-256" id="p-256" id="p-256" id="p-256" id="p-256"

[0256] Mannosylati oncan refer to modification of an amino acid to include a mannose glycoside. Mannosylati oncan occur, e.g., on threonine (T) residues of a polypeptide, e.g., a mini-nucleosom coree protein. To provide one particular example, mono-O-mannosyl glycan s concentrat ine inhibitory GABAergic neurons, and the present disclosure includes mono-0- mannosylation of mini-nucleosome core proteins to target mini-nucleosome core proteins to GABAergic neurons.

Myristoylated Mini-Nucleosome Core Proteins id="p-257" id="p-257" id="p-257" id="p-257" id="p-257"

[0257] Myristoylatio isn a lipid modification that includes covalent linkage of a myristoyl group with an amino acid, e.g., a residue of a polypeptide such as a mini-nucleoso me core protein. Myristoylatio cann occur, e.g., on a glycine (G) residue of a polypeptide such as a mini-nucleosom coree protein. For instanc e,protein N-myristoylation is a lipidic modification that can be present on the alpha-amino group of an N-terminal glycine residue. Myristoylation can contribute to cellula signaling,r protein-protein interaction, and targeti ngof proteins to endomembra neand plasma membrane systems id="p-258" id="p-258" id="p-258" id="p-258" id="p-258"

[0258] In various embodiments, myristoylation of one more residues of a modified mini - nucleosome core protein increases affinity or avidity with a target cell or other binding partner of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the myristoylation modifications. In various embodiments, myristoylation of one more residues of a modified mini-nucleosom coree protein increases affinity or avidity of bindin withg a target receptor of the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the myristoylation modifications. Thus, in some embodiments, myristoylation of one more residue ofs a modified mini-nucleosome core protein increases delivery of a mini-nucleosome core protein, loaded mini-nucleosome core protein, or nucleic acid payload of a mini-nucleosome core protein to a target cell, tissue, or organ. id="p-259" id="p-259" id="p-259" id="p-259" id="p-259"

[0259] In various embodiments, myristoylation of one more residues of a modified mini - nucleosome core protein increases strength (e.g., affinit ory avidity) of association with cell membranes (e.g., endothelial cell membranes) of the modified mini-nucleosome core protein, or loaded mini-nucleosome core proteins including the same, as compared to a referenc minie 107 nucleosome core protein that does not include one or more of the myristoylation modifications.

In various embodiments, myristoylation of one more residues of a modified mini-nucleoso me core protein increases the rate at which the modified mini-nucleosom coree protein, or loaded mini-nucleosom coree proteins including the same, penetrates cell membranes (e.g., endothelial cell membranes) as compared to a referenc mini-e nucleosom coree protein that does not include one or more of the myristoylation modifications. In various embodiments myristoylat, ion of one more residues of a modified mini-nucleosome core protein increases the rate at which the modified mini-nucleosom coree protein, or loaded mini-nucleosome core proteins including the same, enters one or more target cells (i.e., crosses the cell membranes of target cells), such as endothelial cell s,as compared to a referenc mini-nucleosomee core protein that does not include one or more of the myristoylation modifications.

FucosylatedMini-Nucleosome Core Proteins id="p-260" id="p-260" id="p-260" id="p-260" id="p-260"

[0260] Fucosylation is a form of glycosylation that refers to the addition of fucose to an amino acid, e.g., a threonine (T) residue. Fucosylation includes attachment of a fucos eresidu eto N-glycans, O-glycans, and glycolipids.

Pegylated Mini-Nucleosome Core Proteins id="p-261" id="p-261" id="p-261" id="p-261" id="p-261"

[0261] Pegylation (sometimes written PEGylation) includes the modification of biologica moleculesl by covalent conjugation with polyethylene glycol (PEG), a non-toxic, non- immunogenic polymer .PEG is generally understood to be biocompatible and/or to lack immunogenic ity,antigenicity, and/or toxicity. PEG is solubl ine wate rand other organic solvents is ,readily cleared from the body, and has high mobility in solution. Pegylati oncan impact physical and chemical properties of a polypeptide such as a mini-nucleosom coree protein, including properties such as conformation, electrostatic binding, and hydrophobicity.

Pegylation can cause an improvement in pharmacokineti behaviorc of a polypeptide such as a mini-nucleosom coree protein. Pegylation can improve drug solubilit andy decrease s immunogenicity of a polypeptide such as a mini-nucleosome core protein. PEGylation can increases drug stability and/or retention time of a polypeptide such as a mini-nucleosom coree protein in blood. Pegylation can reduce proteolysis and/or renal excretion polypeptide such as a mini-nucleosome core protein, which can reduce the therapeutically effective dose of a 108 polypeptide such as a mini-nucleosome core protein. As those of skill in the art will appreciate, techniques for use of PEG and pegylation in combination with polypeptide agents are well known in the art. The present disclosure includes, withou limt itation, modified mini-nucleoso me core proteins that include a pegylate aminod acid alone or in combination with other modifications of the same or other amino acids of the mini-nucleosom coree protein.

Modified Mini-Nucleosome Core Proteins with Multiple and/or Branched Modifications id="p-262" id="p-262" id="p-262" id="p-262" id="p-262"

[0262] Those of skill in the art will appreciate that a polypeptide such as a mini- nucleosome core protein can include one or more amino acids that each include one or more modifications provide dherein. Accordingly, a mini-nucleosome core protein can include a plurality of modified amino acids each including the same modification, or can include a plurality of modified amino acids of which at least two include different modifications.

Moreover, as those of skill in the art will further appreciate, in some embodiments a single modified amino acid can include two or more modifications (see, e.g., Figs. 20-22). In some embodiments, a single modified amino acid can include two or more modifications, where each of the modifications is covalently linked to the canonical core structure of the amino acid at a different position thereof. In some embodiments, a single modified amino acid can include two or more modifications, where the two or more modifications are present in a modification chain (i.e., a moiety comprising two or more covalently linked modifications, which is itsel covalentlf y linked to an atom of the canonical core structure of the amino acid) . Accordingly, as disclosed herein, an amino acid can be referred to as modified by a particular modification when the canonical core structure of the amino acid is directl covalentlyy linked to the modification and/or when the canonical core structure of the amino acid is indirectly covalently linked to the modification, e.g., when the modification is present in a modification chain and is not directly linked to an atom of the canonical core structure of the amino acid. id="p-263" id="p-263" id="p-263" id="p-263" id="p-263"

[0263] Modification chains of the present disclosure can include a plurality of modifications. In some embodiments, a modification chain includes a plurality of linearly associated modifications, which modifications are directly or indirectly covalently linked in a single linear chain. A modification chain including a single covalently linked linear chain of directl ory indirectly linked modifications can be referred to as an "unbranched" modification chain. Modification chains of the present disclosure can be "branched", e.g., where the 109 modification chain includes a "trunk" that is covalently linked to an atom of the canonical core structure of an amino acid, and which trunk is covalently linked to two or more branches of covalently linked atoms, each of which branches can include one or more modifications. In such branched modification chains, the trunk and two or more branches do not form a single covalently linked linear chain. As provided herein, a trunk or unbranche modificatd ion chain can include any number of modifications provided herein, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications provide dherein, any of which may be the same modification as any other in the trunk or chain or a different modifications from any other in the trunk or chain . As provide d herein, a branch can include any numbe ofr modifications provide dherein, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications provided herein, any of which may be the same modification as any other in the branch or chain or a different modifications from any other in the branch or chain.

Thus, for the avoidance of doubt, a mini-nucleosome core protein, as set forth herein, can include (a) a. nucleic acid binding domain (NABD), and (b) a targeting domain, and in some embodiments can include (a) a nuclei acidc binding domain (NABD), (b) a targeting domain, and (c) a nucleic acid releas edomain, where any of the various mini-nucleosome core proteins provided herein can optional lyinclude one or more modified amino acid residues.

Nucleie Acid Cargos id="p-264" id="p-264" id="p-264" id="p-264" id="p-264"

[0264] Loaded mini-nucleosomes disclosed herein can be loaded with a nucleic acid cargo that is, e.g., RNA, DNA, or a nucleic acid analog thereof A. nucleic acid cargo can be single stranded or double strande d.A nucleic acid cargo can be linear or circular. A nucleic acid cargo can encode one or more of each of a protein, an RNA, an shRNA, an miRNA, an antibody, a nanobody, a Darpin, an Ankyrin repeat, or a polypeptide. For example, a nucleic acid cargo can be a cDNA molecule that encodes at least one functional protein. In various embodiments, a nucleic acid cargo can be an inhibitory RNA, e.g., a gRNA, siRNA, miRNA, or shRNA. id="p-265" id="p-265" id="p-265" id="p-265" id="p-265"

[0265] A nucleic acid cargo can encode, e.g., an RNA, protein, polypeptide, antibody, nanobody, miRNA, shRNA, gRNA, Cas9, non-coding RNA when delivered into a nucleus of any cell. Expression may not be limited to entities mentioned herein. 110 Loaded Mini-Nucleosomes id="p-266" id="p-266" id="p-266" id="p-266" id="p-266"

[0266] A loaded mini-nucleosom ofe the present disclosure can include one or more mini-nucleosom coree proteins of the present disclosure and one or more polynucleoti des.Those of skill in the art will appreciate from the present disclosure that such loaded mini-nucleosomes can be generated from combinin gmini-nucleosom coree proteins and polynucleotides in a variety of ways. Those of skill in the art will appreciate that in, at least one embodimen loadedt, mini-nucleosome assembl ywill occur simply upon inclusion of one or more mini-nucleosome core proteins provided herein and one or more polynucleotides in a solution, e.g., without limitati on,an aqueous solution, e.g., at a standar tempd erature and e.g., vortexing at a standard speed. Methods of generating loaded mini-nucleosom coree proteins therefor includee approaches provided herein and others that will be apparent to those of skill in the art. Those of skill in the art will appreciate that in, at least one embodiment, loaded mini-nucleosome assembly will occur upon inclusion of one or more mini-nucleosom coree proteins provide d herein and one or more polynucleotides in a solution, e.g., without limitati on,an aqueous solution, e.g., at a standard temperature in the presence of catalys tsthat help enhance condensation of nuclei acids.c id="p-267" id="p-267" id="p-267" id="p-267" id="p-267"

[0267] A loaded mini-nucleosom ofe the present disclosure can be at an uncondensed state and a condensed state. A loaded mini-nucleosome is in a condensed state where at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of negative charges in the nucleic acid molecule has been neutralized A loaded. mini-nucleosome is considered in an uncondens stateed when less than 90% of negative charges in the nucleic acid molecule has been neutralized Unless. specified, references to mini-nucleosomes in the present disclosure encompas sat least condensed and uncondens statesed and, where applicable, characteristics thereof. id="p-268" id="p-268" id="p-268" id="p-268" id="p-268"

[0268] A mini-nucleosome can include, e.g., 1 to 10,000 mini-nucleosome core proteins.

A mini-nucleosome can include, e.g., 1 to 100 nucleic acid cargo molecules. id="p-269" id="p-269" id="p-269" id="p-269" id="p-269"

[0269] In some embodiments, loaded mini-nucleosome can be of size between 0.5 to 50 nanometers in diameter. Mini-nucleosomes can include nucleic acid cargo molecules that can have a length of up to 50kb while maintaining a smal ldiameter of between 0.5 and 50nm.

Ill id="p-270" id="p-270" id="p-270" id="p-270" id="p-270"

[0270] In some embodiments, loaded mini-nucleosome can have a molecular weight of between 100 and lOOOOkDa ,e.g., 100, 200, 500, 1000, 3000, 5,000, 8000, 10000 kDa. id="p-271" id="p-271" id="p-271" id="p-271" id="p-271"

[0271] hi various embodiments, loaded mini-nucleosom cane have a. net charge of-100 to 100. In some embodiments, the zeta potent ialof the loaded mini-nucleosome formulation may range from -10 milliVolts to 100 millivolts. In some examples, a complex of nucleic acid cargo and mini-nucleosome core protein is condensed to a minimal size compared to the nucleic acid molecule and polypeptide molecules used to construct the mini-nucleosome particle. The final positive to negative charge ratio is approximately 1:1, thereby forming a non-charged, slightly positively charged or slightly negativel chary ged molecule. The final particle may form in several shapes including rod, spherical or circular but not limited to these. id="p-272" id="p-272" id="p-272" id="p-272" id="p-272"

[0272] In various embodiments, the mini-nucleosom coree protein may be modified with one or more molecul ofes polyethylene glycol of molecular weight of 5 Daltons to 20 kDa. A polyethylene glycol (PEG) moiety maybe attached to any amino acid residu ein the polypeptide. id="p-273" id="p-273" id="p-273" id="p-273" id="p-273"

[0273] In certain embodiments, a loaded mini-nucleosome includes a. ratio of nucleic acid molecules to mini-nucleosome core proteins that is between 1:1 and 1 ;2,000 or betwee n1 ;3 and 1:2,000. In certain embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosome core proteins that is between 1:1 and 1:2,000. In certain embodiments, a loaded mini-nucleosom includese a ratio of nucleic acid molecules to mini- nucleosome core proteins that is betwee n1:1 and 1:1,000, between 1:1 and 1:500, between 1:1 and 1:200, between 1:1 and. 1: 100, between 1:1 and 1:50. In certain embodiments, a loaded, mini-nucleosom includese a ratio of nucleic acid molecul toes mini-nucleosome core proteins that is between 1:3 and 1: 1,000, between 1:3 and 1:500, between 1:3 and 1:200, between 1:3 and 1: 100, or between 1:3 and 1:50. In certain embodiments, a loaded mini-nucleosom includese a ratio of nucleic acid molecules to mini-nucleosome core proteins that is between 1:200 and 1:2,000, between 1:200 and 1:1000, or between 1:200 and 1:500. In certain embodiments, a. loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosome core proteins that is between 1:1 and. 1:50, 1:1 and 1:40, 1:1 and 1:30, 1:1 and 1:20, 1:1 and 1:10, 1:1 and 1:5, 1:1 and 1:4, 1:1 and 1:3, or 1:1 and 1:2. id="p-274" id="p-274" id="p-274" id="p-274" id="p-274"

[0274] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosom coree proteins that, is between 1 nucleic acid molecule to 3 112 mini-nucleosome core proteins (1:3) and 1 nucleic acid molecul toe 3,000 mini-nucleosome core proteins (1:3,000), or within any range there between. id="p-275" id="p-275" id="p-275" id="p-275" id="p-275"

[0275] In various embodiments, a. loaded mini-nucleosome includes a. ratio of nucleic acid, molecules to mini-nucleosom coree proteins that is between 1 nuclei acidc molecule to 3 mini-nucleosom coree proteins (1:3) and 1 nucleic acid molecul toe 2,000 mini-nucleosome core proteins (1:2,000). id="p-276" id="p-276" id="p-276" id="p-276" id="p-276"

[0276] In various embodiments, a. loaded mini-nucleosome includes a. ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nuclei acidc molecule to 3 mini-nucleosom coree proteins (1:3) and 1 nucleic acid molecul toe 1,000 mini-nucleosome core proteins (1:1,000). id="p-277" id="p-277" id="p-277" id="p-277" id="p-277"

[0277] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nucleic acid molecule to 3 mini-nucleosom coree proteins (1:3) and 1 nucleic acid molecul toe 500 mini-nucleosome core proteins (1:500). id="p-278" id="p-278" id="p-278" id="p-278" id="p-278"

[0278] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nucleic acid molecule to 3 mini-nucleosom coree proteins (1:3) and 1 nucleic acid molecul toe 200 mini-nucleosome core proteins (1:200). id="p-279" id="p-279" id="p-279" id="p-279" id="p-279"

[0279] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nucleic acid molecule to 3 mini-nucleosom coree proteins (1:3) and 1 nucleic acid molecul toe 100 mini-nucleosome core proteins (1:100). id="p-280" id="p-280" id="p-280" id="p-280" id="p-280"

[0280] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nucleic acid molecule to 3 mini-nucleosom coree proteins (1:3) and 1 nucleic acid molecul toe 50 mini-nucleosome core proteins (1:50). id="p-281" id="p-281" id="p-281" id="p-281" id="p-281"

[0281] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosome core proteins that is between 1 nucleic acid molecule to 50 mini-nucleosom coree proteins (1:50) and. 1 nucleic acid molecule to 2,000 mini-nucleosome core proteins (1:2,000). 113 id="p-282" id="p-282" id="p-282" id="p-282" id="p-282"

[0282] In various embodiments, a loaded mini-nucleosom includese a ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nuclei acidc molecule to 50 mini-nucleosom coree proteins (1:50) and 1 nucleic acid molecule to 1,000 mini-nucleosome core proteins (1:1,000). id="p-283" id="p-283" id="p-283" id="p-283" id="p-283"

[0283] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nucleic acid molecule to 50 mini-nucleosom coree proteins (1:50) and 1 nucleic acid molecule to 500 mini-nucleosome core proteins (1:500). id="p-284" id="p-284" id="p-284" id="p-284" id="p-284"

[0284] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosome core proteins that is between 1 nucleic acid molecule to 50 mini-nucleosom coree proteins (1:50) and. 1 nucleic acid molecule to 200 mini-nucleosome core proteins (1:200). id="p-285" id="p-285" id="p-285" id="p-285" id="p-285"

[0285] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nuclei acidc molecule to 50 mini-nucleosom coree proteins (1:50) and 1 nucleic acid molecule to 100 mini-nucleosome core proteins (1:100). id="p-286" id="p-286" id="p-286" id="p-286" id="p-286"

[0286] In various embodiments, a loaded mini-nucleosome includes a ratio of nucleic acid, molecules to mini-nucleosom coree proteins that is between 1 nuclei acidc molecule to 200 mini-nucleosom coree proteins (1:200) and 1 nucleic acid molecul toe 2,000 mini-nucleoso me core proteins (1:2,000). id="p-287" id="p-287" id="p-287" id="p-287" id="p-287"

[0287] In various embodiments, a. loaded mini-nucleosome includes a. ratio of nucleic acid, molecules to mini-nucleosom coree proteins that is between 1 nuclei acidc molecule to 200 mini-nucleosom coree proteins (1:200) and 1 nucleic acid molecul toe 1,000 mini-nucleosome core proteins (1:1,000). id="p-288" id="p-288" id="p-288" id="p-288" id="p-288"

[0288] In various embodiments, a. loaded mini-nucleosome includes a. ratio of nucleic acid molecules to mini-nucleosom coree proteins that is between 1 nuclei acidc molecule to 200 mini-nucleosom coree proteins (1:200) and 1 nucleic acid molecul toe 500 mini-nucleosome core proteins (1:500). id="p-289" id="p-289" id="p-289" id="p-289" id="p-289"

[0289] The skilled artisan will appreciate that mini-nucleosome core protein molecul es can be produced and/or constituted by various means, including without limitation in several 114 differe ntsalt conditions including acetate, tri fluoroacetat bicarbonate,e, and chloride. Final formulation of the loaded mini-nucleosom maye be constituted in normal saline, water or any other pharmaceutically acceptable buffers.

Delivery of Loaded Mini-Nucleosomes to Target Cells or Tissues id="p-290" id="p-290" id="p-290" id="p-290" id="p-290"

[0290] In certain embodiments, a mini-nucleosome can deliver a nucleic acid where the target cell is the retinal pigment epithelium (RPE). For efficie ntgene therapy, some embodiments include deliver ofy a large copy numbe ofr genetic cargo such as DNA or RNA into one cell type. For example, in wet-age-related macular degeneratio expressin, ng anti-VEGF in the RPE may provide therapeutic level ofs proteins necessary for inhibiting endothelial cell proliferation and vascular leakage. We provide herein, examples of mini-nucleosomes core proteins (SEQ ID NO. 392) that allow enhanced uptake into the RPE (Figur e10, 11). id="p-291" id="p-291" id="p-291" id="p-291" id="p-291"

[0291] In certain embodiments, a mini-nucleosome can deliver a nucleic acid where the target cell is a neuron in the retina. It has been described that amino acid domain ERE (SEQ ID NO. 156) could be used for enhanced neuronal attachment (Dale D, et al, 1989). We have made use of such domain in a non-viral vector using a GFP construct (SEQ ID NO. 395) with mini - nucleosome core protein (SEQ ID NO. 394) to express GFP to target neuronal cells in the retina (Figur e12). This maybe particularly useful for delivering DNA or RNA to trea tretinal degeneratio causedn by genetic mutations in genes expressed in retinal neurons. id="p-292" id="p-292" id="p-292" id="p-292" id="p-292"

[0292] In various embodiments, a mini-nucleosome can deliver a nucleic acid where the target cell is for e.g. a muscle cell, a liver cell, an endothelial cell, hematopoieti stemc cell, lung epithelial cell,, a pericyte, a beta cell, gut epithelial cell, a microgli alcell, a macrophage cell, a neuronal cell, skin cell, a blood cell, etc. but not limited to these. Various combination of domains described herein (Table 3-12), may allow deliver ofy loaded mini-nucleosomes to certain target cell type for therapeutic effects in other parts of the body including brain, retina, gut, live r,lung, kidney, muscle, pancreas but not limited to it. 115 Pharmaceutical Compositions id="p-293" id="p-293" id="p-293" id="p-293" id="p-293"

[0293] The present disclosure contemplates a "loaded mini-nucleosome therapeutic" that includes a loaded mini-nucleosom ande at least one pharmaceuticall accey ptable carrier.

Formulations of pharmaceutically acceptable carrier solutions are well-known to those skilled in the art, as is the development of suitable dosing and treatment regimens. Typicall y,these formulations can contain 102 genome copies or more of desire dtransgenes. Other factors such as solubilit bioavailability,y, half-life, shelf-life will be contemplated by one skilled in the art. As such, various doses and treatment regiments may be desirable. Loaded mini-nucleosome therapeutic could be used to deliver nucleotides to variety of cell types, tissue types or organs in a human body including retina, liver CNS,, gut etc. but not limited to it. id="p-294" id="p-294" id="p-294" id="p-294" id="p-294"

[0294] A loaded mini-nucleosome therapeutic can be formulated such that it is pharmaceutically acceptable for administrat ionto cells or animals. Loaded mini-nucleosome therapeutic may be administered in vitro, ex vivo or in vivo. A loaded mini-nucleosome therapeutic can be administered to a subject either alone or in combination with one or more other therapeutic modalities, e.g., antibodies, steroids, vitamins AAVs, etc. id="p-295" id="p-295" id="p-295" id="p-295" id="p-295"

[0295] In certain instances, a loaded mini-nucleosome therapeut canic include one or more nuclei acidc cargos that each or togethe encoder one or more distinct expression products . id="p-296" id="p-296" id="p-296" id="p-296" id="p-296"

[0296] In certain circumstances, it will be desirable to deliver the loaded mini - nucleosome formulations in suitably formulat pharmaceuticaled compositions disclosed herein either by subcutaneous, intraocular, intravitre parenteral, al, intravenous, intramuscular, intrathecal, topical oral,, intraperitoneal injections, or by nasal inhalation but not limited to these techniques. Solutions of the loaded mini-nucleosome formulations may be prepared in sterile water, sterile saline and may also suitably mixed with one or more surfactants, such as pluronic acid. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof. Storage preparations may contain preservatives to prevent microorganisms from growing. id="p-297" id="p-297" id="p-297" id="p-297" id="p-297"

[0297] A suitable means of administrat ionof a loaded mini-nucleosome therapeutic agent can be selected based on the condition or disease to be treated and upon the age and condition of a subject. Dose and method of administration can vary depending on the weight age,, condition, and the like of a patient, and can be suitably selected as needed by those skilled in the art. 116 id="p-298" id="p-298" id="p-298" id="p-298" id="p-298"

[0298] In various instances, a loaded mini-nucleosome therapeut agentic composition can be formulated to include a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers includ withoute, limitati on,any and all solvents disper, sion media, coatings, antibacter ialand antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Compositions of the present invention can include a pharmaceutically acceptable salt e.g.,, an acid additio saltn or a base addition salt. id="p-299" id="p-299" id="p-299" id="p-299" id="p-299"

[0299] In various embodiments, a composition including a loaded mini-nucleoso me therapeutic agent as described herein, e.g., a sterile formulation for injection, can be formulated in accordance with conventional pharmaceutical practices using distill watered for injection as a vehicle. For example, physiological saline or an isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride may be used as an aqueous solution for injection, optionall iny combination with a suitable solubilizing agent, for example, alcohol such as ethanol and polyalcohol such as propylen glycole or polyethylene glycol and, a nonionic surfactant such as polysorbate 80™, HCO-50 and the like. id="p-300" id="p-300" id="p-300" id="p-300" id="p-300"

[0300] As disclosed herein, a loaded mini-nucleosom there apeut agentic composition may be in any form known in the art. Such forms include, e.g., liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposome ands suppositories. id="p-301" id="p-301" id="p-301" id="p-301" id="p-301"

[0301] Selection or use of any particular form may depend in, part, on the intended mode of administration and therapeutic application. For example, compositions containing a composition intended for systemic or local delivery can be in the form of injectab leor infusib le solutions. Accordingl y,a loaded mini-nucleosome therapeutic agent composition can be formulate ford administrat ionby a parenteral mode (e.g., intravenous, subcutaneous, intraperiton eal,or intramuscular injection). As used herein, parenteral administrat ionrefers to modes of administrat ionother than enteral and topical administrati on,usuall byy injection, and include, without limitation, intravenous, intranasal intr, aocular pulmonar, y,intramuscular , intraarteri al,intrathecal, intracapsul ar,intraorbit al,intracardiac, intradermal, intrapulmonar y, intraperiton eal,transtracheal, subcutaneous, subcuticular, intraarticul subcapsularar, , subarachnoid, intraspinal, epidural, intracerebral, intracrania intracl, arotid and intrasterna l injection and infusion. 117 id="p-302" id="p-302" id="p-302" id="p-302" id="p-302"

[0302] A parenteral route of administration can be, for example, administrat ionby injection, transnasal administration, transpulmonar adminisy tration, or transcutaneous administrati on.Administration can be systemic or local by intravenous injection, intramuscul ar injection, intraperitoneal injection, subcutaneous injection. id="p-303" id="p-303" id="p-303" id="p-303" id="p-303"

[0303] In various embodiments, a loaded mini-nucleosom therapeutice agent composition of the present invention can be formulat edas a solution, microemulsion, dispersion, liposome, or other ordered structur suitablee for stable storage at high concentrati on.Sterile injectable solutions can be prepared by incorporating a composition described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerat ed above, as required, followed by filt ersterilizatio n.Generally, dispersions are prepared by incorporating a composition described herein into a sterile vehicle that contain as basic dispersion medium and the required other ingredients from those enumerat edabove. In the case of sterile powders for the preparation of sterile injectable solutions, methods for preparation include vacuum drying and freeze-drying that yield a powder of a composition described herein plus any addition desireal dingredient (see below) from a previously sterile-filter soluted ion thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithi n,by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition a reagent that delays absorption, for example, monostearate salts, and gelatin. id="p-304" id="p-304" id="p-304" id="p-304" id="p-304"

[0304] A loaded mini-nucleosom there apeutic agent composition can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in wate ror another pharmaceuticall accey ptable liquid. For example, the loaded mini - nucleosome therapeut agentic composition can be formulat byed suitably combinin gthe therapeutic molecule with pharmaceuticall accey ptable vehicles or media, such as sterile water and physiological saline vegetable, oil, emulsifier suspension, agent surf, actant, stabilizer, flavoring excipient, diluent, vehicle, preservative binder, followe, byd mixing in a unit dose form required for generally accepte dpharmaceutic alpractices. The amount of loaded mini - nucleosome therapeut agentic included in the pharmaceutic alpreparations is such that a suitable dose within the designated range is provided. Nonlimiting examples of oily liquid include 118 sesame oil and soybean oil, and it may be combine dwith benzyl benzoate or benzyl alcohol as a solubilizing agent. Other items that may be included are a buffe suchr as a phosphate buffer or, sodium acetate buffer a, soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol or phenol, and an antioxidant. The formulat injectioned can be packaged in a suitable ampule. id="p-305" id="p-305" id="p-305" id="p-305" id="p-305"

[0305] In some embodiments, a loaded mini-nucleosom therapeutice agent composition can be formulat fored storag eat a temperature below 0°C (e.g., -20°C or -80°C). In some embodiments, the composition can be formulate ford storage for up to 2 years (e.g., one month, two months three, months, four months, five months six, months, seven months eight, months, nine months ,10 months ,11 months ,1 year, 11/2 years, or 2 years) at 2-8°C (e.g., 4°C). Thus, in some embodiments, the compositions described herein are stable in storage for at least 1 year at 2-8°C (e.g., 4°C). id="p-306" id="p-306" id="p-306" id="p-306" id="p-306"

[0306] In particular instances, a loaded mini-nucleosom there apeut agentic composition can be formulat edas a solution. In some embodiments, a composition can be formulated, for example, as a buffere solutiond at a suitable concentration and suitable for storage at 2-8°C (e.g., 4°C). id="p-307" id="p-307" id="p-307" id="p-307" id="p-307"

[0307] Compositions including a loaded mini-nucleosom therapeutice agent as described herein can be formulated in immunoliposome compositions. Such formulations can be prepared by methods known in the art. Liposomes with enhanced circulation tim eare disclosed in, e.g., U.S. Pat. No. 5,013,556. id="p-308" id="p-308" id="p-308" id="p-308" id="p-308"

[0308] In certain embodiments, compositions can be formulat withed a carrier that will protect the compound against rapid release, such as a controlled release formulati on,including implants and microencapsulated deliver systemy s. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides polyglycolic, acid, collagen , polyorthoester ands, polylactic acid. Many method fors the preparation of such formulations are known in the art. See, e.g., J. R. Robinson (1978) "Sustained and Controlled Release Drug Delivery Systems", Marcel Dekker, Inc., New York. id="p-309" id="p-309" id="p-309" id="p-309" id="p-309"

[0309] In some embodiments compositions, can be formulat ined a composition suitable for intrapulmon aryadministration (e.g., for administration via an inhaler or nebulizer) to a mammal such as a human. Methods for formulating such compositions are well known in the 119 art. Dry powder inhaler formulations and suitable system sfor administrat ionof the formulations are also known in the art. Pulmona ryadministrat ionmay be oral and/or nasal. Examples of pharmaceutic aldevice fors pulmonary delivery include metered dose inhalers dry, powder inhalers (DPIs), and nebulizers. For example, a composition described herein can be administered to the lungs of a subject by way of a dry powder inhaler .These inhalers are propellant-fre devicese that deliver dispersible and stable dry powder formulations to the lungs.

Dry powder inhalers are well known in the art of medicine and include, without limitati on:the TURBOHALER® (AstraZeneca; London, England) the AIR® inhaler (ALKERMES@; Cambridge, Mass.); ROTAHALER® (GlaxoSmithKline London,; England); and ECLIPSETM (Sanofi-Aventis Paris; , France). See also ,e.g., PCT Publication Nos. WO 04/026380, WO 04/024156, and WO 01/78693. DPI devices have been used for pulmonary administration of polypeptides such as insulin and growth hormone. In some embodiments, a composition described herein can be intrapulmonarily administered by way of a metered dose inhaler .These inhalers rely on a propellant to deliver a discrete dose of a compound to the lungs. Additional devices and intrapulmonar administraty ionmethods are set forth in, e.g., U.S. Patent Application Publication Nos. 20050271660 and 20090110679, the disclosures of each of which are incorporated herein by referenc ine thei rentirety. id="p-310" id="p-310" id="p-310" id="p-310" id="p-310"

[0310] In some embodiments, loaded mini-nucleosom there apeut agentic compositions can be formulat fored deliver toy the eye, e.g., in the form of a pharmaceutically acceptabl e solution, suspension or ointment. A preparation for use in treating an eye can be in the form of a sterile aqueous solution containing, e.g., addition ingredieal nts such as, but not limited to, preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, and viscosity-increasi ngagents. A preparation as described herein can be administered topically to the eye of the subject in need of treatment (e.g., a subject afflict edwith AMD) by conventional methods, e.g., in the form of drops, or by bathing the eye in a therapeutic solution, containing one or more compositions. id="p-311" id="p-311" id="p-311" id="p-311" id="p-311"

[0311] A variety of devices for introducing drugs into the vitrea cavityl of the eye may be appropriate, in certain embodiments, for administrat ionof a composition as described herein.

For example, U.S. Publication No. 2002/0026176 describes a pharmaceutical-containin plugg that can be inserted through the sclera such that it projects into the vitreous cavity to deliver the 120 pharmaceutic alagent into the vitreous cavity. In another example, U.S. Patent No. 5,443,505 describes an implantabl device efor introduction into a suprachoroidal space or an avascular region for sustained release of drug into the interior of the eye. U.S. Patent Nos. 5,773,019 and 6,001,386 each disclose an implantable drug deliver devicey attachable to the scleral surfac eof an eye. Additional method ands devices (e.g., a transscleral patch and deliver viay contact lenses for) deliver ofy a loaded mini-nucleosom therapeutice agent to the eye are described in, e.g., Ambat iand Adamis (2002) Prog Reti nEye Res 21(2):145-151; Ranta and Urtti (2006) Adv Drug Delivery Rev 58(11): 1164-1181; Barocas and Balachandran (2008) Expert Opin Drug Delivery 5(1): 1-10(10); Gulse nand Chauhan (2004) Inves Opthalmolt Vis Sci 45:2342-2347; Kim et al. (2007) Ophthalmic Res 39:244-254; and PCT publication no. WO 04/073551, the disclosures of which are incorporated herein by referenc ine thei rentirety. id="p-312" id="p-312" id="p-312" id="p-312" id="p-312"

[0312] In various embodiments, subcutaneous administration can be accomplished by means of a device, such as a syringe, a prefille syringe,d an auto-injector (e.g., disposable or reusable), a pen injector a, patch injector, a wearable injector, an ambulato rysyringe infusion pump with subcutaneous infusion sets, or other devic efor subcutaneous injection. id="p-313" id="p-313" id="p-313" id="p-313" id="p-313"

[0313] In some embodiments, a loaded mini-nucleosom therapeutice agent composition described herein can be therapeutically deliver edto a subjec tby way of local administration. As used herein, "local administration" or "local deliver" y,can refer to deliver thaty does not rely upon transport of the loaded mini-nucleosome therapeut agentic composition or loaded mini - nucleosome therapeut agentic to its intended target tissue or site via the vascula systr em. For example, the loaded mini-nucleosome therapeutic agent composition may be deliver edby injection or implantati ofon the composition or agent or by injection or implantation of a devic e containing the composition or agent. In certain embodiments, following local administration in the vicinity of a target tissue or site, the composition or agent, or one or more component s thereof, may diffuse to an intended target tissue or site that is not the site of administration. id="p-314" id="p-314" id="p-314" id="p-314" id="p-314"

[0314] In some embodiments the, compositions provide dherein are present in unit dosage form, which unit dosage form can be suitable for self-administration. Such a unit dosage form may be provided within a container typical, ly, for example, a vial cartr, idge, prefilled syringe or disposable pen. A doser such as the doser device described in U.S. Pat. No. 6,302,855, may also be used, for example, with an injection system as described herein. 121 id="p-315" id="p-315" id="p-315" id="p-315" id="p-315"

[0315] A suitable dose of a loaded mini-nucleosome therapeutic agent composition described herein, which dose is capable of treating or preventing a disorder in a subject can, depend on a variet yof factors including, e.g., the age, sex, and weight of a subject to be treated, the condition or disease to be treated and, the particular loaded mini-nucleosome therapeutic agent used. Other factors affecting the dose administered to the subject include, e.g., the type or severit ofy the condition or disease .Other factors can include, e.g., other medica ldisorders concurrently or previously affecting the subject, the genera healthl of the subject the, genetic disposition of the subject, diet, time of administration, rate of excretion, drug combination, and any other additional therapeutic thats are administered to the subject. It should also be understood that a specific dosage and treatment regime nfor any particular subject can also be adjuste based d upon the judgment of a medical practitioner. id="p-316" id="p-316" id="p-316" id="p-316" id="p-316"

[0316] A loaded mini-nucleosome therapeutic agent solution can include a therapeutically effective amount of a composition described herein. Such effective amounts can be readily determined by one of ordinary skill in the art based, in part, on the effe ctof the administered composition, or the combinatori aleffect of the composition and one or more additional active agents, if more than one agent is used. A therapeutically effective amount can be an amount at which any toxic or detrimental effects of the composition are outweighed by therapeutically beneficial effects. id="p-317" id="p-317" id="p-317" id="p-317" id="p-317"

[0317] Pharmaceutical forms of loaded mini-nucleosome therapeutic formulations suitable for injection can include sterile aqueous solutions or dispersions. A formulation can be sterile and must be fluid to allow proper flow in and out of a syringe. A formulation can also be stable under the conditions of manufacture and storage. A carrier can be a solvent or dispersion medium containing, for example, wate rand saline or buffered aqueous solutions. Preferably, isotonic agents, for example, sugars or sodium chloride can be used in the formulations. For human administration, final preparations and compositions should meet sterilit pyroy, genicit y, and the general endotoxin levels safety, and purity standards as required by the US FDA and EU regulatory standards Temperature. and exposure to other proteins can alter the properties of loaded mini-nucleosomes. The final preparations and compositions must be stored at appropriate temperature preferabls, aty 2-8 degree Celsius or at room temperatur (20-25e degree Celsius). 122 id="p-318" id="p-318" id="p-318" id="p-318" id="p-318"

[0318] In addition, one skilled in the art may also contemplate addition deliveryal method may be via electroporation, sonophoresis, intraosseous injections methods or by using gene gun. Vectors may also be implanted into microchips, nano-chips or nanoparticles. id="p-319" id="p-319" id="p-319" id="p-319" id="p-319"

[0319] In certain embodiments, the compositions described herein may be formulate ind a kit. Such kits may be used for therapeutic or diagnost purposes.ic The present disclosure provides, among other things, one or more compositions togethe withr one or more pharmaceutically-acceptabl excipiee nts, carriers, diluents, adjuvants, and/or other components, as may be employe ind the formulation of a composition consisting of mini-nucleosome core proteins and nuclei acids,c and in the preparation of therapeut agentsic for administration to a mammal, and in particularly, to a human, for one or more diseases described herein. In particular such, kits may include one or more of the disclosed mini-nucleosom coree protein compositions in combination with instructions for using nucleic acids in the treatment various disorders in a mammal, and may typically include containers prepared for convenient commercial packaging. id="p-320" id="p-320" id="p-320" id="p-320" id="p-320"

[0320] Compositions described herein can be administered to an animal that is a mammal, e.g., a human. Compositions described herein are also applicable to animals of commercial interes livestoct, andk, household pets such as dogs and cats .Compositions in kits can include partiall ory significantly purifie loadedd mini-nucleosomes compositions, either alone or, in combination with one or more other ingredients or drugs for therapeutic or diagnosti c use. Therapeutic kits can also be prepared that include at least one loaded mini-nucleoso me component based gene therapy compositions disclosed herein and instructions for using the composition as a therapeutic agent. The container means for such kits may typically include at least one vial test, tube, flask, bottle syringe, or other containe means,r into which the disclosed mini-nucleosomes composition(s) may be placed, and preferably suitably aliquoted. 123 Applications id="p-321" id="p-321" id="p-321" id="p-321" id="p-321"

[0321] Mini-nucleosomes provide dherein can, in various embodiments, be characterized by small size, ability to enter cells by receptor mediated or passive diffusio procesn ses, precision in the location of gene expression, precision in the duration of gene expression, and/or retention until releas eof nucleic acids in the cytoplasm of the nucleus of a target cell. Some of the desired application of the mini-nucleosome technology are described herein. id="p-322" id="p-322" id="p-322" id="p-322" id="p-322"

[0322] The present disclosure furthe includesr the recognition that modified mini - nucleosomes of the present disclosur cane target deliver ofy nuclei acidc payloads to particul ar cells or tissues and/or to achieve expression of an expression product encoded by a nucleic acid payload in one or more particular cells or tissues. As will be readily apparent from the present disclosure, deliver and/ory expression of a nucleic acid payload in a particular cell or tissue can be useful in a variety of methods and compositions that benefit from the presence of a particul ar expression product (e.g., a particular protein) in a particular targeted cell or tissue. id="p-323" id="p-323" id="p-323" id="p-323" id="p-323"

[0323] In certain embodiments, a modified mini-nucleosom ofe the present disclosure includes one or more phosphorylate residuesd (e.g., where one or more, or all, of the phosphorylated residues are selected from serine, threonin e,or tyrosine residues). The present disclosur includese the recognition that the presence, rather than the specific number or position, of the phosphorylated residu emediate thes ability of the modified mini-nucleosom to etarget delivery of nucleic acid payloads to particular cells or tissue and/ors to achieve expression of an expression product encoded by a nucleic acid payload in one or more particular cells or tissues.

Without wishing to be bound by any particular scientif theory,ic the present disclosure includ es that at least certain advantages that characterize phosphorylated mini-nucleosome core proteins (e.g., targeti ngof particular cell or tissues) result from interactio ofn the modification with cell surfac ereceptors of target cells and are therefor note sequenc specie fic or specific to the position of any modified residue. The present disclosure specifical lyincludes the recognition that phosphorylated mini-nucleosome core proteins can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, target cells that are cells of the centr alnervous system (CNS). In certain embodiments, a phosphorylated mini-nucleosom coree proteins can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, CNS neurons. In certain embodiments, a phosphorylate mini-d nucleosom coree proteins can deliver a 124 nucleic acid payload to, and/or cause expression of a nucleic acid payload in, CNS astrocytes, microglia, oligodendrocytes, or glia. In certain embodiments, a phosphorylated mini - nucleosome core protein can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, spinal cord cells, e.g., spinal cord neuron ors spinal cord glial cell s.In various embodiments, expression of an expression product encoded by a nucleic acid payload associated with a modified mini-nucleosome core protein (e.g., a phosphorylated mini - nucleosome core protein, e.g., in a loaded mini-nucleosome) is increased as compared to expression achieved under referenc (e.g.,e same or similar) conditions using a referenc (e.g.,e unmodified) mini-nucleosome core protein. The present disclosure recognizes that expression in various such target cell types is significant for treatment of certain conditions that effe ctor are effected by target cell types, e.g., diseases of the CNS. id="p-324" id="p-324" id="p-324" id="p-324" id="p-324"

[0324] In certain embodiments, deliver ofy a nucle icacid payload and/or expression of a nucleic acid payload in target cells is facilitat byed a particular route of deliver y.In various embodiments in which a phosphorylate mini-d nucleosom coree protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discloss ed herein, administration to a subject is by a route that achieves delivery to cells of the central nervou system.s In various embodiments in which a phosphorylated mini- nucleosome core protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discls osed herein, administrat ionto a subject is by a route that achieve sdeliver toy CNS neurons. In various embodiments in which a phosphorylated mini-nucleosome core protein deliver a snuclei acidc payload to, and/or cause expression of a nuclei acidc payload in, one or more target cell s,cell types, or tissues disclosed herein, administration to a subjec tis by a route that achieves deliver toy CNS astrocytes, microglia, oligodendrocytes, or glia. In various embodiments in which a phosphorylated mini- nucleosome core protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discls osed herein, administrat ionto a subject is by a route that achieve sdeliver toy spinal cord cells, e.g., spinal cord neurons or spinal cord glial cell s.Those of skill in the art will be familiar with routes of administrat ionto particular cells, cell types, and tissues. In certain particular embodiments, administrat ionto a subject is by injection, e.g., injection to the CNS (e.g., to a cell or tissue of the CNS). In certain 125 particular embodiments, administration to a subject is intrathecal, intracrania orl, intra-cisterna magna. In certain particular embodiments, administrat ionto a subject is intrathecal. id="p-325" id="p-325" id="p-325" id="p-325" id="p-325"

[0325] In various embodiments, a phosphorylate mini-d nucleosom coree protein can include multiple phosphorylated residue s,e.g., one, two, three, or four phosphorylated residues .

In various embodiments, a phosphorylate mini-d nucleosom coree protein can include one or more amino acid modifications disclosed herein that is not phosphorylation. Accordingl y,the present disclosure includes combinations of different modifications present in a single modified mini-nucleosome core protein. A phosphorylated amino acid of a modified mini-nucleoso me can be present in any domain or at any amino acid position of a modified mini-nucleosome A . phosphorylated amino acid of a modified mini-nucleosome can be present, without limitation, in a linker domain or targeting domain. id="p-326" id="p-326" id="p-326" id="p-326" id="p-326"

[0326] In certain embodiments, a modified mini-nucleosom ofe the present disclosure includes one or more sulfate residuesd (e.g., where one or more, or all, of the sulfate residud es are selected from serine, threonine, or tyrosine residues) .The present disclosure includes the recognition that the presence, rather than the specific number or position, of the sulfated residue mediates the ability of the modified mini-nucleosome to target deliver ofy nucleic acid payloads to particular cells or tissues and/or to achieve expression of an expression product encoded by a nucleic acid payload in one or more particular cells or tissues. Without wishing to be bound by any particular scientif theoryic ,the present disclosure includes that at least certain advantages that characterize sulfate mini-nucleosomed core proteins (e.g., targeti ngof particular cell or tissues) result from interaction of the modification with cell surface receptors of target cells and are therefor note sequenc specife ic or specific to the position of any modified residue. The present disclosure specifical lyincludes the recognition that sulfate mini-nucd leosome core proteins can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, target cells that are cells of the centr alnervous system. In certain embodiments, a sulfate d mini-nucleosom coree protein can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, CNS neurons. In certain embodiments, a sulfate mini-d nucleosom coree protein can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, CNS astrocytes, microglia, oligodendrocyt ores, glia. In certain embodiments, a sulfated mini-nucleosom coree protein can deliver a nucleic acid payload to, and/or cause expression of a 126 nucleic acid payload in, spinal cord cells, e.g., spinal cord neuron ors spinal cord glial cell s.In various embodiments, expression of an expression product encoded by a nucleic acid payload associated with a modified mini-nucleosome core protein (e.g., a sulfate mini-nucleosomed core protein, e.g., in a loaded mini-nucleosome) is increased as compared to expression achieved under referenc (e.g.,e same or similar) conditions using a referenc (e.g.,e unmodified) mini- nucleosome core protein. The present disclosure recognize thats expression in various such target cell types is significant for treatment of certain conditions that effect or are effected by target cell types, e.g., diseases of the CNS. id="p-327" id="p-327" id="p-327" id="p-327" id="p-327"

[0327] In certain embodiments, deliver ofy a nucle icacid payload and/or expression of a nucleic acid payload in target cells is facilitat byed a particular route of deliver y.In various embodiments in which a sulfate mini-nucleosomed core protein deliver a snuclei acidc payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discloss ed herein, administration to a subjec tis by a route that achieves deliver toy cells of the centr alnervou systs em. In various embodiments in which a sulfate mini-nucd leosome core protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discls osed herein, administrat ionto a subject is by a route that achieve sdeliver toy CNS neurons. In various embodiment ins which a sulfated mini- nucleosome core protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discls osed herein, administrat ionto a subject is by a route that achieve sdeliver toy CNS astrocytes, microglia, oligodendrocyt ores, glia. In various embodiments in which a sulfate mini-nucleosomed core protein deliver a s nucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discls osed herein, administrat ionto a subject is by a route that achieves delivery to spinal cord cells, e.g., spinal cord neuron ors spinal cord glial cell s.Those of skill in the art will be familiar with routes of administration to particular cell s,cell types, and tissues. In certain particular embodiments, administrat ionto a subject is by injection, e.g., injection to the CNS (e.g., to a cell or tissue of the CNS). In certain particular embodiments, administration to a subject is intrathecal, intracranial or, intra-cister namagna. In certain particular embodiments, administration to a subject is intrathecal. 127 id="p-328" id="p-328" id="p-328" id="p-328" id="p-328"

[0328] In various embodiments, a sulfated mini-nucleosome core protein can include multiple sulfate residued s,e.g., one, two, three, or four sulfated residues. In various embodiments, a sulfat edmini-nucleosom coree protein can include one or more amino acid modifications disclosed herein that is not sulfation. Accordingl y,the present disclosure includ es combinations of different modifications present in a single modified mini-nucleosome core protein. A sulfate aminod acid of a modified mini-nucleosome can be present in any domain or at any amino acid position of a modified mini-nucleosome A .sulfate aminod acid of a modified mini-nucleosom cane be present, without limitati on,in a linker domain or targeting domain. id="p-329" id="p-329" id="p-329" id="p-329" id="p-329"

[0329] In certain embodiments, a modified mini-nucleosom ofe the present disclosure includes one or more acetylated residue (e.g.,s where one or more, or all, of the acetylated residues are lysine residues) .The present disclosure includes the recognition that the presence, rather than the specific number or position, of the acetylated residue mediates the ability of the modified mini-nucleosome to target delivery of nucleic acid payloads to particular cells or tissue and/ors to achieve expression of an expression product encoded by a nucleic acid payload in one or more particular cells or tissues. Without wishing to be bound by any particul ar scientif theoryic ,the present disclosure includes that at least certain advantages that characterize acetylated mini-nucleosome core proteins (e.g., targeting of particular cell or tissues) result from interaction of the modification with cell surfac ereceptors of target cells and are therefor note sequenc specie fic or specific to the position of any modified residue. The present disclosure specifical lyincludes the recognition that acetylated mini-nucleosome core proteins can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, target cells that are CNS neurons and/or retinal cells. In certain embodiments, an acetylated mini-nucleosome core protein can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, CNS neurons. In certain embodiments, an acetylated mini-nucleosome core protein can deliver a nucleic acid payload to, and/or cause expression of a nucle icacid payload in, retinal cells, e.g., retinal neuron includings one or more of photoreceptor bipolars, cells, retinal ganglion cells, horizontal cells and amacrine cells). In certain embodiments, an acetylated mini- nucleosome core protein can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, photoreceptor cells (e.g., rods and/or cones). In various embodimen ts, expression of an expression product encoded by a nucleic acid payload associated with a 128 modified mini-nucleosom coree protein (e.g., an acetylated mini-nucleosome core protein, e.g., in a loaded mini-nucleosome) is increased as compared to expression achieved under reference (e.g., same or similar) conditions using a reference (e.g., unmodified) mini-nucleosom coree protein. The present disclosure recognizes that expression in various such target cell types is significant for treatment of certain conditions that effect or are effected by target cell types, e.g., diseases of the CNS, including diseases of the retina (e.g., Retiniti pigments osa and/or Stargardt’s disease). id="p-330" id="p-330" id="p-330" id="p-330" id="p-330"

[0330] In certain embodiments, deliver ofy a nucle icacid payload and/or expression of a nucleic acid payload in target cells is facilitat byed a particular route of deliver y.In various embodiments in which an acetylated mini-nucleosom coree protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discloss ed herein, administration to a subject is by a route that achieves delivery to CNS neurons and/or retinal cell s.In various embodiments in which an acetylated mini- nucleosome core protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discls osed herein, administrat ionto a subject is by a route that achieve sdeliver toy CNS neurons. In various embodiments in which an acetylated mini-nucleosome core protein deliver a snucleic acid payload to, and/or cause expression of a nuclei acidc payload in, one or more target cell s,cell types, or tissues disclosed herein, administration to a subjec tis by a route that achieves deliver toy retinal cells, e.g., retinal neurons including one or more of photoreceptor bipolars, cell s,retinal ganglion cells, horizontal cells and amacrine cells). In various embodiments in which an acetylated mini-nucleosom coree protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discls osed herein, administrat ionto a subject is by a route that achieve sdelivery to photoreceptor cells (e.g., rods and/or cones) . Those of skill in the art will be familiar with routes of administrat ionto particular cells, cell types, and tissues. In certain particular embodiments, administrat ionto a subject is by injection, e.g., injection to the eye (e.g., to a cell or tissue of the eye). In certain particular embodiments, administration to a subject is intravitre supracal, horoida l,or subretinal.

In various embodiments, an acetylated mini-nucleosom coree protein can include multipl e acetylated residue s,e.g., one, two, three, or four acetylated residues. In various embodiments, an 129 acetylated mini-nucleosome core protein can include one or more amino acid modifications disclosed herein that is not acetylation. Accordingl y,the present disclosure includ es combinations of different modifications present in a single modified mini-nucleosome core protein. An acetylated amino acid of a modified mini-nucleosome can be present in any domain or at any amino acid position of a modified mini-nucleosome An. acetylated amino acid of a modified mini-nucleosome can be present, without limitati on,in a linker domain or targeting domain. id="p-331" id="p-331" id="p-331" id="p-331" id="p-331"

[0331] In certain embodiments, a modified mini-nucleosom ofe the present disclosure includes one or more mannosylated residues (e.g., where one or more, or all, of the mannosylated residues are serin eresidues) .The present disclosure includes the recognition that the presence, rather than the specific number or position, of the mannosylated residue mediates the ability of the modified mini-nucleosome to target deliver ofy nuclei acidc payloads to particular cells or tissue and/ors to achieve expression of an expression product encoded by a nucleic acid payload in one or more particular cells or tissues. Without wishing to be bound by any particul ar scientif theoryic ,the present disclosure includes that at least certain advantages that characterize mannosylat mini-ed nucleosom coree proteins (e.g., targeting of particular cell or tissues) result from interaction of the modification with cell surfac ereceptors of target cells and are therefor e not sequenc specie fic or specifi cto the position of any modified residue. The present disclosure specifical lyincludes the recognition that mannosylated mini-nucleosome core proteins can deliver a nucleic acid payload to, and/or cause expression of a nucle icacid payload in, target cells that are CNS neurons and/or retinal cells. In certain embodiments, a mannosylated mini - nucleosome core protein can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, CNS neurons. In certain embodiments, a mannosylat mini-ed nucleosome core protein can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, retinal cell s,e.g., retinal neuron includings one or more of photoreceptor bipolars, cells, retinal ganglion cells, horizontal cells and amacrine cells). In certain embodiments, a mannosylated mini-nucleosome core protein can deliver a nucleic acid payload to, and/or cause expression of a nucleic acid payload in, photoreceptor cells (e.g., rods and/or cones). In various embodiments, expression of an expression product encoded by a nucleic acid payload associated with a modified mini-nucleosome core protein (e.g., a 130 mannosylat mini-ed nucleosom coree protein, e.g., in a loaded mini-nucleosome) is increased as compared to expression achieved under referenc (e.g.,e same or similar) conditions using a referenc (e.g.,e unmodified) mini-nucleosom coree protein. The present disclosure recognizes that expression in various such target cell types is significant for treatment of certain conditions that effect or are effected by target cell types, e.g., diseases of the CNS, including diseases of the retina (e.g., Retiniti pigments osa and/or Stargardt’s disease. id="p-332" id="p-332" id="p-332" id="p-332" id="p-332"

[0332] In certain embodiments, deliver ofy a nucle icacid payload and/or expression of a nucleic acid payload in target cells is facilitat byed a particular route of deliver y.In various embodiments in which a mannosylated mini-nucleosome core protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discloss ed herein, administration to a subject is by a route that achieves delivery to CNS neurons and/or retinal cell s.In various embodiments in which a mannosylated mini - nucleosome core protein deliver a snucleic acid payload to, and/or cause expression of a nucleic acid payload in, one or more target cells, cell types, or tissue discls osed herein, administrat ionto a subject is by a route that achieve sdeliver toy CNS neurons. In various embodiments in which a mannosylated mini-nucleosome core protein deliver a snucleic acid payload to, and/or cause expression of a nuclei acidc payload in, one or more target cell s,cell types, or tissues disclosed herein, administration to a subjec tis by a route that achieves deliver toy retinal cells, e.g., retinal neurons including one or more of photoreceptor bipolars, cell s,retinal ganglion cells, horizontal cells and amacrine cells). In various embodiments in which a mannosylated mini-nucleosome core protein deliver a snuclei acidc payload to, and/or cause expression of a nuclei acidc payload in, one or more target cells, cell types, or tissue discloss ed herein, administration to a subject is by a route that achieve sdelivery to photoreceptor cells (e.g., rods and/or cones). Those of skill in the art will be familiar with routes of administrat ionto particular cells, cell types, and tissues.

In certain particular embodiments, administrat ionto a subject is by injection, e.g., injection to the eye (e.g., to a cell or tissue of the eye). In certain particular embodiments, administration to a subject is intravitre supracal, horoida l,or subretinal. id="p-333" id="p-333" id="p-333" id="p-333" id="p-333"

[0333] In various embodiments, a mannosylated mini-nucleosom coree protein can include multiple mannosylated residue s,e.g., one, two, three, or four mannosylated residues. In various embodiments, a mannosylated mini-nucleosome core protein can include one or more 131 amino acid modifications disclosed herein that is not mannosylation. Accordingl y,the present disclosur includese combinations of different modifications present in a single modified mini - nucleosome core protein. A mannosylated amino acid of a modified mini-nucleosom cane be present in any domain or at any amino acid position of a modified mini-nucleosome. A mannosylat edamino acid of a modified mini-nucleosom cane be present, without limitation, in a linker domain or targeting domain.

Gene Therapy id="p-334" id="p-334" id="p-334" id="p-334" id="p-334"

[0334] In various embodiments, mini-nucleosomes provided herein can be used in methods of gene therapy. The general principles of gene therapy are well known in the art and include the deliver ofy a polynucleotide to a subject in need thereof to provide an expression product (e.g., an mRNA, protein, or inhibitory RNA) of therapeutic value. In some embodiments, gene therapy can include gene or protein replaceme nttherapy (e.g., enzyme replaceme nttherapy) ,augmentati oron, target inhibition. In various embodiments, mini- nucleosomes provide dherein can be applied to rescue deleterious effects of any mutations that cause diseases including, without limitation, Cystic fibrosis, Duchenne muscular dystrophy, Stargardt’s disease, Age-relat edmacular degenerati on,Huntingt Hemophion, lia A, Spinal muscular atrophy, Usher syndrome etc. In such diseases, a genetic mutation renders a gene nonfunctional or not available. In such cases, replacing the mutated gene by a functional copy may be benefici alto the patients. By incorporating a functional cDNA or whole gene into a loaded mini-nucleosome and, delivering it to desire dcells or tissues, one may receive, in various embodiments, a therapeutic benefit. id="p-335" id="p-335" id="p-335" id="p-335" id="p-335"

[0335] In some embodiments, mini-nucleosomes provided herein can be applied to inhibit genes that are upregulated and disease causing. For example, P53 overexpression has been described in various diseases. In some instances, it is also beneficial to knock down genes at specific cells or tissue tos downregulate genes that cause inflammation, hypoxia etc. to have therapeutic effects. 132 Ex-vivo Engineered Cells id="p-336" id="p-336" id="p-336" id="p-336" id="p-336"

[0336] Mini-nucleosomes of the present disclosure can be used to engineer cells ex vivo.

Cells can be engineered to express therapeut icsin various ways. One such cell is immune cell, e.g., T cell. Immune cells can be genetically engineered to express new proteins or receptors that may allow immune recognition of cancerous cells or other harmful cell types for killing and clearance. Such genetic engineering may be performed ex vivo. In various embodiments, mini- nucleosomes provided herein can be used in methods of genetically engineering cells ex vivo.

Combination of domains provide dherein, may allow loaded mini-nucleosome entry to variet yof T cells and deliver a genetic cargo to the nucleus in such cell s.The genetic cargo may encode and/or allow expression of chimeric receptors, knockdown of genes or other therapeutic entity .

Such cells may then be infused into patient fors therapy. One skilled in the art, may contempl ate using loaded mini-nucleosomes for creating chimeric antigen receptor T cells (CAR T cells) for use in immunotherapy. id="p-337" id="p-337" id="p-337" id="p-337" id="p-337"

[0337] In some embodiments, mini-nucleosomes provided herein can be applied to engineering stem cells ex vivo to expres snew proteins or receptors for therapeutic purposes.

Combination of domains provide dherein, may allow loaded mini-nucleosome entry to variet yof stem cells to deliver a genetic cargo to the nucleus/cytoplasm in such cell s.The genetic cargo may allow expression of chimeric receptors, knockdown of genes or other therapeutic entity .

Such cells may then be infused into patient fors therapy. One skilled in the art, may contempl ate using loaded mini-nucleosomes for creating chimeric stem cells or chimeric hematopoieti stemc cells for use in immunotherapy.

Gene Editing And Base Excision Repair id="p-338" id="p-338" id="p-338" id="p-338" id="p-338"

[0338] Gene editing, base editing and manipulat ionis also an applicable area for this mini-nucleosome technology described herein. Gene editing and base excision repair are state- of-the-ar techt nolog iesthat allow correcting a genet mutationic or editing the genes at the DNA or RNA level Towards. this application, a loaded mini-nucleosom maye incorporat enucleic acids that encode for gRNA, sgRNA, spCas9, saCas9, dCas9, cytidine deaminase and several other enzymes that help cleave DNA or convert one base to another. One skilled in the art can appreciate that incorporating multiple gRNAs and Cas9 or similar editing enzymes in an AAV is 133 a cumbersome and often inefficie procent ss. Hence, using the method and compositions described herein, that enable easys compaction of nucleic acids onto loaded mini-nucleosomes allows incorporation of several gRNAs and even the largest of Cas9 genes to deliver to desired cells.

Antibody Delivery id="p-339" id="p-339" id="p-339" id="p-339" id="p-339"

[0339] Antibodies are a class of drugs that have been life changing for millions of patien tsworldwide. However, one big drawback in this therapy is the requirement of repeat administration which poses immense burde nto patients, physicians and caregivers One. skilled in the art can appreciate that a DNA molecule can be used to express antibodies. Mini- nucleosome technology described herein, provides an opportunity to vectoriz thee antibody and deliver to desired cells in the patien tsto create a long-te rmdepot in their bodies to reduce the burde nof multiple administrati on.These DNA molecules that express part or whole of antibody domains can be incorporated into loaded mini-nucleosomes to create a long term therapeuti c option for patien tsthat take antibody drugs. One skilled in the art may also vectorize and deliver other antibody like molecules such as nanobody, antibody mimetics, fusion peptides, antibody fragments cameli, dor cameli dsingle-domain antibody fragmen usingts mini-nucleosome core proteins.

Vaccine Delivery id="p-340" id="p-340" id="p-340" id="p-340" id="p-340"

[0340] Genetical engineely red DNA or RNA can produce an antigen to provide a protective immunological response. Nucleic acid vaccines have several potential advantages such as wide-range immunological response over conventional vaccines. Mini-nucleosome technology described herein, can incorporate and deliver such DNA or RNA constructs to desired cells or tissue ins animals including humans to protect from several viral, bacterial or parasitic infections. 134 Cosmetics id="p-341" id="p-341" id="p-341" id="p-341" id="p-341"

[0341] Genetically engineered DNA or RNA can be developed for several cosmeti c application fors example to enhance muscle mass, repair skin in burn victims, for weight loss, to improve immune function, to slow aging and many other applications. Mini-nucleosome technology described herein, can incorporate and deliver applicable DNA or RNA constructs to desired cells or tissue ins animals including humans for desired cosmeti ceffect. id="p-342" id="p-342" id="p-342" id="p-342" id="p-342"

[0342] In various embodiments, the present disclosure furthe providesr vectors relatin tog preventing or treating a disease in humans or other animals. A prophylactically or therapeutically effective amount of a composition could be administered via intravenous, intramuscular, intranasal intrap, eriton eal,subcutaneous, intracerebral subret, inal, intravitre viaal, lumbar puncture topical, rectal,, or direct delivery to local organs or tumors but not limited to these techniques. The composition includes of nucleic acid complexes, each complex consisting essentially of a single or more nucleic acid molecul ande one or more mini-nucleosome core protein molecules. id="p-343" id="p-343" id="p-343" id="p-343" id="p-343"

[0343] The present disclosure provides, among other things, improve dmethods of condensing DNA, RNA and thei ranalogs etc. for efficie ntdeliver intoy human cells to treat certain diseases and/or cosmetic applications. The nucleic acid deliver edmay also have application tos deliver vaccines.

EXAMPLES Example 1: Design and synthesis of mini-nucleosome core proteins id="p-344" id="p-344" id="p-344" id="p-344" id="p-344"

[0344] This Example is representati ofve methods and compositions relating to mini - nucleosome core proteins. In this Example, amino acid sequences of peptides (that can condense nucleic acids into loaded-mini-nucleosomes) and their synthesis process are described. id="p-345" id="p-345" id="p-345" id="p-345" id="p-345"

[0345] Loaded mini-nucleosomes of the present Example are produced for efficie ntgene transf erand release of loaded nucleic acid cargo to various cell types. Loaded mini-nucleosomes of the present Example are designed to actively engage with cell surfac evia binding to cell surfac eproteins to, be translocat toed the cytoplasm/nucleus in cells, and to allow release of the nucleic acid cargo. These characteristics can be achieved by mini-nucleosome core protein and 135 loaded mini-nucleosomes designe based d on structured protein/DNA interaction. Accordingly, the present Example includes mini-nucleosom coree proteins that include one or more amino acid domains that enhance one or more of cellular attachment, enhanced uptake, enhanced stability, active transport to the nucleus of a target cell, and release via peptidases. id="p-346" id="p-346" id="p-346" id="p-346" id="p-346"

[0346] In the present Example, synthesized mini-nucleosome core proteins may include, without limitation, a sequence according to any one of SEQ ID NOS: 389-394, or other sequences derived from domains disclosed herein in Table 3-12, or any combination thereof .

Mini-nucleosome core proteins of the present Example are peptides with net positive charge >8 at pH 7 and isoelectr pointic >9. For example, SEQ ID NO: 394 is a mini-nucleosome core protein sequence including multiple DNA bindin gdomains (KRHRK) combined with multipl e Neuronal attachment domains (ERE) and a poly-Argini nedomain (RRRRR). In this same construct, Leucine (L)s surround the poly-Argini nedomain to separate charged domains with hydrophobic amino acids, enabling the cell attachment targeting domain to bind to the cell surface. In this construct, the mini-nucleosome core protein (SEQ ID NO: 394) is designed for enhanced attachment to neurons via ERE domain while the poly-Arginine domain would help cell entry. The present Example also includes mini-nucleosome core proteins with various linkers positioned between certain domains, and examples of linker includes those provided in SEQ ID NOS: 389-394. By design, KRH in SEQ ID NO: 394 also serves as a cut site for PCSK1 for enhanced release of nucleic acids. Other nucleic acid releas edomains or cleavag domainse that could be include ind mini-nucleosom coree proteins includ withoute, limitati on,those described in Table 9. Domains for inclusion in mini-nucleosom coree proteins can also be derived for other peptidases including, withou limitationt those in Table 9. id="p-347" id="p-347" id="p-347" id="p-347" id="p-347"

[0347] Mini-nucleosome core proteins of the present Example including, mini- nucleosome core proteins according to SEQ ID NOS: 389-394, include various combinations of sequenc feate ures that allows efficie ntcondensation with nucleic acid molecules and deliver ofy loaded mini-nucleosomes to desired cell types, e.g., animal cells and tissues. In certain mini- nucleosome core proteins of the present Example, an oligomerization domain is included in a mini-nucleosom coree protein in order to cause a loaded mini-nucleosom coree protein formed by association of the mini-nucleosom coree protein with a nuclei acidc cargo to have a relative ly smaller size as compared to a referenc loadede mini-nucleosom coree protein, e.g., as compared 136 to a loaded mini-nucleosom incle uding mini-nucleosom coree proteins that lack the oligomerization domain(s) but otherwis eare identical in amino acid sequence. Exemplary oligomerization domains include those provided in Table 11. Similarly, endosomal entry and escape signal mays also be included in mini-nucleosome core proteins for enhanced stability and release. id="p-348" id="p-348" id="p-348" id="p-348" id="p-348"

[0348] Mini-nucleosome core proteins of the present Example can be synthesized by various methods. One method of synthesizing mini-nucleosom coree proteins is peptide synthesi s.Peptide synthesis allows linking of amino acids via amide bonds. For example, mini- nucleosomes core proteins can be chemicall synthesizedy via a condensation reaction between carboxyl group of one amino acid to the amino group of the next desired amino acid, in order of the sequenc ofe a mini-nucleosom coree protein. An established method of peptide syntheses is known in the art as solid phase peptide synthesis. id="p-349" id="p-349" id="p-349" id="p-349" id="p-349"

[0349] Several strategi escan optionally be applied to protect the amino (N-terminal) and carboxy-termina (C-tl erminal of) mini-nucleosom coree proteins of the present disclosure. If the mini-nucleosom coree protein is lyophilized, the lyophilize peptided may contain traces of salts used during the synthesis process. Other method ofs mini-nucleosom coree protein production include expressing the mini-nucleosome core protein in a cell system or in vivo form DNA constructs encoding the mini-nucleosome core protein. Produced mini-nucleosome core proteins can be purified by a variety of methods known in the art. For instance, several resins may be utilized during the process. Mini-nucleosom corees proteins in, various instances of the present Example, are >90% pure. However, a less pure <90% core protein may also be used to form a loaded mini-nucleosome Mini-. nucleosom corees proteins in, various instances of the present Example, are >90% conjugated with PEG. However, a less conjugated (<90%) or non- conjugated core protein may also be used to form a loaded mini-nucleosome Mini-nucleosomes. core protein purity can be determined by high-pressure liquid chromatography (HPLC) and identity confirmed by mass spectromet tory the very least.

Example 2. Production of loaded Mini-nucleosomes id="p-350" id="p-350" id="p-350" id="p-350" id="p-350"

[0350] This Example describes technique relatins tog production of a loaded mini - nucleosome, including without limitation a loaded mini-nucleosome of Example 1. Loaded mini 137 nucleosomes of the present Example include a nucleic acid cargo (DNA or RNA) condensed with mini-nucleosome core proteins with net positive charges. The mini-nucleosome core protein net positive charge neutralizes negativ chargese of the nucleic acid cargo, resulting in nanometer sized particles. Conjugation of the said mini-nucleosomes core proteins and DNA or RNA can occur in small or large quantitie Theres. are 2 phosphates meaning 2 negative charges associated with ever ybase. The present Example provides that at least 90% of DNA negative charges are neutralized by a nucleosome core protein positive charge. For example, 90-95 percent of DNA negative charges need to be neutralized for efficient condensation of the nucleic acids with a mini-nucleosome core protein. Various mini-nucleosome core proteins of the present Example can include amino acid domains that enhance one or more of cellula r attachment, cellul uptake,ar protein stability, active transport to the nucleus of a target cell, and releas eof nuclei acidc cargo. Thus, certain mini-nucleosom coree proteins provide dherein can be particularly useful in certain contexts. During the process of mixing the nucleic acids and mini-nucleosomes core proteins to produce a loaded mini-nucleosome, the mixture of nucleic acids and mini-nucleosome core proteins can be mixed or vortexed between lOOrpm sto 4000rpms. In the process of conjugation of nucleic acids, certain catalysts, such as NaOH and spermidines, that enhance the condensation reaction may be added. These catalys tscan be added to the reactor prior to adding the polypeptides and nucle icacids. The nucleic acids may be added in concentratio rangingns from 0.1 microgram/microlit toer lOOgrams/liter. Mini-nucleosom es core proteins may be added at a concentration of 0.1 microgram/microliter to lOOgrams/liter. The nucleic acids may be added at once or may be added gradually, e.g., steadily or in sequentially in drops to a vortexing solution. Once the mixing is over, the condensed material i.e.,s, loaded mini-nucleosomes may be allowed to be equilibrated for a period of several minutes to several hours, e.g., a period of 2 minutes to a period of 6 hours, prior to purification. Dialysis may be performed to remove impurities and exchange buffers at this stage. Loaded mini-nucleosomes may be purified using several techniques. One such technique is to centrifuge the particles at high speed in a column with molecular weight cutoff parameter ofs 1 kiloDalton or higher. The centrifugation speed may range from 7000 xg to 10,000 xg depending on the sample volume.

Similarl y,duration of centrifugation may vary from 20 minutes at room temperatur toe one hour depending in sample volume. Another technique available to purif ythe mini-nucleosomes is 138 dialysis The. purification techniqu maye not be limited to these two techniques and those of skill in the art will be aware of various further purificatio techn nique froms literature that can be used to purif yprotein/nucl acideic complexes. Finall y,the loaded mini-nucleosomes may be eluted or collected in endotoxin free water, norma lsaline or any other buffered solution but not limited to these The. expected recovery of DNA is -30-70%. Loaded mini-nucleosomes may also undergo further centrifugation in molecula weightr cut-of columnsf to further concentrat thee amount of vector genome in the solution. In the present Example the, loaded mini-nucleosome is formulate tod minimize the presence of endotoxi n.Typical sources of endotoxin are known to include plasmids, peptide synthesis, or from materials used in the prep. Hence, endotoxin free plasmid cans be used, and materials and equipment that have been scrubbed of endotoxin can be used, during preparations described in this Example. id="p-351" id="p-351" id="p-351" id="p-351" id="p-351"

[0351] A bioreactor can also be used to formulate loaded mini-nucleosomes for consistent mixing of the nucleic acids and peptides to produce particles for commercial and clinical use.

Example 3: Favorable shapes/sizes and formulations for loaded mini-nucleosomes id="p-352" id="p-352" id="p-352" id="p-352" id="p-352"

[0352] Provided in this Example are techniques to produce loaded mini-nucleosomes in various formulations, including formulations useful for administration to cells and to mammalian subjects e.g.,, humans. Loaded mini-nucleosomes can be formulate tod different shape and/or sizes parameter bases d on the mini-nucleosom coree protein amino acid sequence and the buffer conditions in which the synthesis occurs. Loaded mini-nucleosomes can be formulated in different conditions, e.g., with solubilit suitabley for therapeut use.ic Solubility of loaded mini- nucleosomes in wate rand/or norma lsaline is one means to allow non-toxi formulc ation of compositions for administration to patients, and to ease of deliver intoy patients. To form loaded mini-nucleosomes represented in Figure 7, core proteins were synthesized by solid phase synthesis using trifluoroacetate buffers. 200 micrograms of DNA (SEQ ID NO: 396) were added to 1 milligra ofm lyophilized core proteins and vortexed together and, purified to produce loaded mini-nucleosomes (Figur e7). Buffe exchanr ge was performed and final formulation of mini- nucleosome was made in sterile endotoxin, free water. 1 microgram of each kind of mini- nucleosomes was dilut edin water and then place don grids that were stained with freshly 139 prepared in 0.75% uranyl acetate in methano solutionl for two minutes. Grids were dipped in 100% ethanol and then blotted into lens absorbent paper .The grids were then air-dried for few minutes with film side up and taken for imaging with Hammatsu ORCA HR camera (Figur e7).

The polynucleotide utilized in generating loaded mini-nucleosome core proteins of the present disclosur ase a plasmid encoding luciferase, but those of skill in the art will appreciate that the present Example is broadly demonstrative of the genera capacitl y of mini-nucleosome core proteins of the present disclosure to associate with polynucleotides and form loaded mini - nucleosomes. Luciferase plasmi dis representati ofve nucleic acid in general, including, without limitati on,plasmids, linear nucleic acids, RNA and DNA of all kinds .In other cases, e.g., RNA or DNA of other sequences or structures could be used in producing loaded mini-nucleosomes.

Luciferase plasmid condensed with core protein of SEQ ID NO: 393, led to spiral/helical-shaped loaded mini-nucleosome (Figur e7A). Luciferase plasmid condensed with core protein with SEQ ID NO: 390, led to rod/lobul shapedar loaded mini-nucleosomes (Figure 7B). A mixture of circular and rod like molecules were observed for loaded mini-nucleosome produced by condensation of luciferase plasmid with core protein SEQ ID NO: 391 (Figur e7C). There are other buffe conditir ons and amino acid sequenc withe varying charge and iso-electr pointic that could produce spherical or circular loaded mini-nucleosomes Molec. ules of different shapes and sizes can enhance tropism to certain cell types. Differently shaped viruses transduce different cell types more effectively. For example, the tobacco mosaic virus is a rod/helical shaped nucleocapsid structure that transduces tobacco plant cell s,HIV is round or ball-shaped that infects white blood cells, and AAV2 is an icosahedral shape that transduces liver cells effectively. We observed better transduction tropism of spiral shaped mini-nucleosomes compared to rod shaped ones in muscle cells (Figur e18). We have been able to formulate different shapedly loaded mini-nucleosomes as described herein. Distinct mini-nucleosomes can also be purified based on unique shapes and sizes. 140 Example 4: Route of administration- Intravenous (systemic) and application in systemic diseases such as Hemophilia A. id="p-353" id="p-353" id="p-353" id="p-353" id="p-353"

[0353] This Example demonstrate thats loaded mini-nucleosomes can be deliver edby intravenous routes to express proteins in the liver and other organs. Balb/c mice were restrained using standard techniques and insulin syringes were used to deliver loaded mini-nucleosomes and plasmid control vias tail vein injections. F8 expressing plasmid constructs ("F8 plasmi"d; see, e.g., MN #1 and MN #2, figur e8) were prepared by condensation of SEQ ID NO: 390 and SEQ ID NO: 391, respectively, with F8 plasmid DNA (SEQ ID NO: 460). Plasmid sequenc for e GFP expressing construct is provided in SEQ ID NO: 8. In the present Example, to target loaded mini-nucleosomes to liver cell s,we incorporated 2 NGR amino acid domains alongside nucleic acid bindin gdomains (SEQ ID NO: 3). NGR domains in AAV2 have been shown to promote aVp5 integr inbinding. NGR domains are implicated in heparan sulfate binding, known as receptor for AAV2. AAV2 is known for high liver tropism. KRH amino acid motif also incorporated in these core proteins serve as a cut site for PCSK1 for enhanced release of nucleic acids. Inclusion of multiple KRH amino sequences should enhance release of loaded mini - nucleosomes. Each mouse received 40 micrograms dose of either MN #1, MN #2 or naked plasmid F8 (SEQ ID NO: 460). To test for expression of F8 protein, ~150pl blood was collected by cheek bleed technique before (1 day prior) and after treatments (post treatme nt-3 days, 1 week, 2 weeks, 1 month 3, months and 4 months) Serum. was prepared from blood using standard techniques. F8 Elisa (Aviva Systems Biology) was performed according to manufacturer’s instructions using 1:6 serum dilutions. Loaded mini-nucleosomes #1 (MN #1 includes SEQ ID NO: 390 + F8 plasmid) and MN #2 (MN #2 includes SEQ ID NO: 391 + F8 plasmid) expressed approximately six folds more F8 compared the level of F8 detected by ELISA in pre-treatme samplesnt MN. #1 sustained significantly elevated level ofs expression at 3 months and 4 months afte ar single injection of loaded mini-nucleosom (Figure e8). Control mice treated with naked plasmid encoding F8 (not complexe withd mini-nucleosome core proteins) did not demonstrate significant increase in F8 expression at either time points (Figure 8)• id="p-354" id="p-354" id="p-354" id="p-354" id="p-354"

[0354] In another experiment, direct GFP fluorescence was observed in tissue collecteds from mice that underwent intravenous injection of loaded- mini-nucleosomes carrying GFP 141 expressing plasmid (SEQ ID NO: 390 + GFP plasmid, SEQ ID NO: 395) (Figur e9). Briefly, mice were perfused with lx PBS and sacrificed. Entir eliver was collected following dissection.

The liver tissues were fixed in 4% paraformaldehyde overnight then washed in IxPBS, immerse d in 15% sucrose for few hours and then in 30% sucrose solution overnight for cry opreservation.

The tissue weres then placed in a plasti vialc and frozen using OCT compound for sectioning. -micron thick tissue sections were obtained using a cryotome. The liver sections were mounted with mounting media with or without DAPI, coversli pand sealed. Images were acquired by Leica SP5 confocal and epi-fluorescent scopes. id="p-355" id="p-355" id="p-355" id="p-355" id="p-355"

[0355] Result demonss trated that when deliver edby intravenous route mini, - nucleosomes successfull reacy hed liver and mini-nucleosome cargo-encoded genes were expressed in liver cells (Figur e9). Expression in multiple liver cell types was observed. The observations of the present Example suggest that delivery of loaded mini-nucleosomes to liver is not dependent upon targeting domains. One of skill in the art, in view of the data provide din the present Examples, would understand that loaded mini-nucleosomes can be deliver edto cells in kidney and spleen via intravenous delivery since,, like the live r,these organs normall y function in clearance of, e.g., drugs. id="p-356" id="p-356" id="p-356" id="p-356" id="p-356"

[0356] One example of a condition that in, view of the present disclosure, can be treated by use of a loaded mini-nucleosome therapeut agentic is Hemophilia A. Hemophilia A is a sever ebleeding disorder caused by mutation in factor 8, a clotting factor. It is inherited in an X- lined recessive manner. It occurs in approximately 1 in 5,000 live births. Most serious implications are internal bleeding that may lead to death. Severit dependsy on amount of F8 circulating in the body. 75% of the hemophilia patien tstake a recombinant F8 product as therapy. Subjects receiving F8 therapy are repeatedly infused intravenously, leading to huge burde nfor patients, physicians, and caregiver overs time. Currently, gene therapy trials are underway to deliver long term expression of F8 via AAVs. However, F8 is a large gene that cannot be fully incorporated in AAV. Thus, mini-F8 has been utilized to deliver functional domains of F8 to trea thist disease It. is well known that mini-F8 doesn’t have the same functional capability and stability as of full-length F8. Moreover, 20-40 % of population already has neutralizing antibodies against AAV that will render a large population of Hemophilic patien tsunable to receive the AAV-based medicine. In addition, if a further treatment were to be 142 needed afte ar first discontinued course of AAV treatme nt,AAV vector cannots be redosed due to immunogenic ity.By being able to deliver full size of F8 gene (Figur e8) and because of its redosable nature (Figur e17), loaded mini-nucleosomes solve these two problems of AAV gene therapy. Thus, the present disclosure provides techniques to deliver loaded mini-nucleosom es into different cell types in the systemic space such as live r,kidney, spleen etc. using intravenous mode of delivery, for use in many conditions of which Hemophilia A is exemplary. id="p-357" id="p-357" id="p-357" id="p-357" id="p-357"

[0357] Other systemic diseases that often stem from defects in secrete proteinsd could also be treated using loaded mini-nucleosomes therapeut agents.ic The present Example (Figure 8) demonstrated that loaded-mini-nucleosomes, deliver edintravenou sly(systemic administration), produce proteins at levels higher than the therapeutic threshold which is approximately 10% of endogenous level determineds by various clinical trials demonstrati ng, among other things, therapeut potentic ialof mini-nucleosomes as therapeut agentsic for treatment of, e.g., systemic diseases where a secreted protein can be expressed by variet yof cell types. In some cases, expression can be restricted to certain cell types by using a cell-type specific promoter. One skilled in the art would also understand from the present disclosure that other tissue suchs as brain ,heart, muscles etc. may also be accessed and transduce viad intravenous deliver They. targeting mechanism built into the mini-nucleosome core proteins shall aid in that context. id="p-358" id="p-358" id="p-358" id="p-358" id="p-358"

[0358] When injected intravenously, loaded mini-nucleosomes may be deliver edat a dose greater than le5 genome copies per kg and up to a dose of 1625 copies per kg of body weight (e.g., at about le5, le6, le7, le8, le9, lelO, 1615, le20, or 1625 copies per kg body weight, or any range there between). Volume of the material may range from 1- 900 milliliter s (e.g., 1, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 millilite Thers). loaded mini-nucleosomes may also be administered repeatedly (e.g., a selected volume and/or numbe ofr genome copies can be administered multiple time sor divided among two or more does). 143 Example 5: Route of administration- Intraocular id="p-359" id="p-359" id="p-359" id="p-359" id="p-359"

[0359] This example demonstrate thats loaded mini-nucleosomes can be deliver edby intra-ocula router to express proteins in the retinal pigment epithelium (RPE) or in other retinal neurons such as photoreceptors, bipolar cells and ganglion cell s.In the present Example, Balb/c mice were anesthetized by IP injection with Ketamine/Xylazi (90-100ne mg/kg +10 mg/kg) and positioned underneath a microscope. Mice eyes were dilated with topical Tropicamide (1%) and lul of loaded mini-nucleosomes (total dose 1.5 micrograms in mice) were injected into the vitreous cavity using 32 gauge blunt needle passing through the incision made by a 25-gauge needle below the limbus. At various time points, mice were perfused withlOml of IxPBS, and then sacrificed using standar techniques.d Mice were enucleated and eyecups were collected and incubated in 4% paraformaldehyde overnight. The eyecups were washed with IxPBS, then immerse din 15% sucrose for few hours and then in 30% sucrose solution overnig htfor cryopreservation. The eyecups were then placed in a plasti vialc and frozen using OCT compound for cryo-sectionin g.10-micron thick tissue sections were obtained for staining. The retinal sections were mounted with mounting media with or without DAPI, coversli pand sealed.

Image swere acquired by Leica SP5. For wholemount imaging, eyecup swere fixed in 4% paraformaldehyde overnight. Eyecups were washed in IxPBS, retina was removed and the remaining eyecup or RPE wholemount was processed for staining. The RPE tissue was wholemounted with mounting media, coversli pand sealed. Images were acquired by Leica SP5.

Native GFP fluorescenc weree observed in retina and RPE cells (Figur e10, 11 & 12). id="p-360" id="p-360" id="p-360" id="p-360" id="p-360"

[0360] To target the RPE cells, the present Example utilized a mini-nucleosom coree protein (SEQ ID NO: 392) that could bind to the phagocytic proteins like MERTK. RPE are phagocytic cells, that extend thei rmicrovilli to the photoreceptor inner/outer segment junction.

MERTK is expressed in those microvilli. In SEQ ID NO: 392, we incorporated the "eat me" signal ass descried in Table 8. In literature, "eat me" signal ares described as domains exposed in cellula debrisr that are primed for phagocytosis (Wei Li, Journal of Cell physiology, 2016, which is incorporated herein by reference). To the present invent’sor knowledg thesee, "eat me" signal s have never been utilized in the contex oft non-viral vector before.s These "eat me" signal domains have not been previously applied for non-viral vector tos target the RPE cells. 144 id="p-361" id="p-361" id="p-361" id="p-361" id="p-361"

[0361] To selectiv elytransduce photoreceptor thes, present Example utilized core proteins like those of SEQ ID NO:394. SEQ ID NO:394 included a neuron alattachment element (ERE) described herein Table 8, that could allow transduction into ganglion cells, bipolar cells and photoreceptors which are all neurons in the retina (Figur e12). This neuronal attachment domain has not been previously applied for non-viral vector tos target neurons. The present disclosur providese that this neuron altargeted vector can transduce neurons in the brain via local or systemic administratio Then. present disclosure furthe provider fors targeting photoreceptor bindin gand internalization by incorporating lectin binding domains (described in Table 4) in mini-nucleosomes for attachment to photoreceptor extracellular matri xto enhance uptake An. integr inbinding domain incorporated in the mini-nucleosome core protein (SEQ ID NO: 390) also could transduc RPEe cells in rat eyes exclusively when deliver edintraocular (Figur e11).

Moreover ,more than one domain could be utilized to selectiv elytransduc ae plurality of diverse cell types. This core protein (SEQ ID NO: 390) with integr inbindin gproperties may also be utilized for deliver ofy nucleic acids to other cell types that express high level ofs aV|35 integrin.

The present disclosure further provide uses of other intra-ocula injectionr technique suchs as subretin al,suprachoroida l,intra-cameral, or topical administration to target photoreceptor RPE,s, Mueller cells or other cell types in the retina. id="p-362" id="p-362" id="p-362" id="p-362" id="p-362"

[0362] Provided herein are technique tos deliver loaded mini-nucleosomes into differe nt cell types in the retina using intravitre supracal, horoida l,or subretinal mode of deliver Diseasy. es like retinal degeneration are mostly caused by mutations in genes expressed in the photoreceptors. Age-related macular degeneration (AMD), is a disease of retinal pigment epithelium (RPE) and choriocapillaries that, affects >10 million Americans and >100 million people worldwide, Currently, the predominant technology to deliver gene therapy vector tos photoreceptors and RPE is a surgical techniqu wheree viruses are injected subretinally into the retina. However, subretinal procedure is a complex surger yperformed in the operating room by a trained Ophthalmic surgeon. There is an unmet need at least in that in, the United states, there are only a handful of surgeons trained to perform this surgery. One way to reduce the burde nfor patien tsand physicians is to develop vector thats can be injected intravitreally that can pass through the retina to transduce the photoreceptors and RPE. Intravitre injectial on can be performed by all ophthalmologist in an in-patient visit. Loaded mini-nucleosom therae py solves 145 this problem as intravitreal injections could transduce photoreceptors and RPE selectively (Figur e10, 11 and 12). This makes mini-nucleosomes highly suitable for treating most retinal diseases with genetic defects. id="p-363" id="p-363" id="p-363" id="p-363" id="p-363"

[0363] When injected intraocular, the loaded mini-nucleosomes may be deliver edat a dose greater than le5 genome copies per eye and up to a dose of 1625 copies per eye (e.g., at about le5, le6, le7, le8, le9, lelO, 1615, le20, or 1625 copies per, or any range there between).

Volume of the material may range from 10- 500 microliters when injected subretinally (e.g., 1, 5, , 20, 30, 40, 50, 100, 200, 300, 400, or 500 microliters) and 10-250 microliters when injection is intravitre supracal, horoidal, or intracameral (e.g., 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, or 250 microliters) A. loaded mini-nucleosome therapeutic agent may also be administered repeatedl y (e.g., a selected volume and/or numbe ofr genome copies can be administered multiple time sor divided among two or more does).

Example 6: Route of administration- Intranasal id="p-364" id="p-364" id="p-364" id="p-364" id="p-364"

[0364] This example demonstrate thats loaded mini-nucleosomes can be deliver edby intra-nas alroute to express proteins in lung, trachea, and gut cell s.In the present Example to, target epithelial cells in the lung epithelium, 2 NGR amino acid domains were included in a mini-nucleosom coree protein alongside nuclei acidc binding domains (see use of NGR amino acid domains SEQ ID NO: 390). To the present inventor’s knowledg NGRe, domains have never been utilized to create and deliver non-viral DNA/protein complexes to retinal cells as disclosed herein. NGR domains in AAV2 have been shown to promote a.Vp5 integr inbinding .

NGR domains are implicated in heparan sulfate binding, known as receptor for AAV2. id="p-365" id="p-365" id="p-365" id="p-365" id="p-365"

[0365] In the present Example, Balb/c mice were anesthetized by IP injection with Ketamine/Xylazine (90-100 mg/kg +10 mg/kg) and the anesthetized mice were positioned underneath a microscope for visual of the nasal area for intranasal delivery lul, of loaded mini - nucleosome (SEQ ID NO: 390 + GFP plasmid) solution was delivered into the nasal cavity ever y few seconds until 12 microliters were deliver edto each nasal side. Total dose of 25 micrograms was delivere Followingd. sacrifice, mice lung was processed to obtain 1 Omi cron thick sections.

Sections were washed in PBS and incubated in blocking buffe (0.1%r TritonX-100, 1% BSA, 3% donkey serum) for Ihr and then incubated in CFTR antibody (prepared on blocking buffer) 146 blocking buffer overnight at 4 degree Celsius Next. day wash in PBS 3x5 min and incubated in AlexaFlour-555 (Donkey Anti-rabb itIgG secondary) in blocking buffe atr RT for !hour and washed in PBS 3x5 min. Mounting media was added and coversli pwas applied and sealed.

Native fluorescence of GFP was obtained in the 486nm channel of Leica SP5 scope in the 486- nm wavelength and CFTR expression in the 555-nm channel. We observed loaded mini- nucleosomes expression as early as 3 days and at PI-3 months as well (Figur e13). We observed expression in the epithelium of both alveo liand bronchioles (Fig 13A and 13C) depicted by sharp green fluorescen alongce with CFTR staining. Co-localization of CFTR and GFP (Fig 13C) demonstrate expressis on of genes encoded by mini-nucleosomes in lung epithelium. Higher magnificati imageson taken from an alveoli ring (Figures 14 A, B and C) also clearly exhibit bright green ring of GFP fluorescen ince the epithelium together with red fluoresc ine CFTR stained cells. id="p-366" id="p-366" id="p-366" id="p-366" id="p-366"

[0366] In the present Example, whole lung tissue and biodistribution via mini- nucleosome was also evaluated (Figur e15). Whole lung tissue was extracted form mice following perfusion and sacrifice. Lung tissue was fixed in 4% PF A and washed with IxPBS.

Whole tissue weres placed in the Odyssey imager for detecting GFP native fluorescence.

Uninjected control did not exhibit any fluorescence (Figur e15). Loaded mini-nucleosomes including plasmid nucleic acid cargo encoding GFP demonstrated GFP fluorescence in whole lung tissue in 5-week post injection samples (Figur e15). id="p-367" id="p-367" id="p-367" id="p-367" id="p-367"

[0367] Provided herein are technique tos deliver loaded mini-nucleosomes into differe nt cell types in tissues of the pulmonary space such as lung epithelium, and/or trachea using intranasal mode of deliver Geneticy. diseases such as cystic fibrosis affect the lung and other organs. To deliver genes to the lung, the intranasal is one of the routes of choices. We observed that loaded-mini-nucleosomes when deliver edintranasally, expresse sproteins in the alveo liand bronchioles (Figur e13). These are tissues that would normall exprey ss the CFTR protein implicated in cystic fibrosis. In other diseases, this route of administration can be used to produce therapeutic proteins that could alleviate other diseases. Intranasal route may also provide access to other organs such as the gut and brain (Figure 16). Inclusion of NGR domains in the mini-nucleosom coree proteins (SEQ ID NO: 390), allowed enhanced uptake and release of DNA molecules into the nucleus for high level ofs sustained expression. This is evidenc edin 147 Figure 16 by the bright green fluorescen obserce ved from loaded-mini-nucleosomes vs no such pattern in the untreated animals (lung image in the first row in Figure 16) at 5-weeks post treatment. We also observed transducti ofon expression of GFP in tracheal epithelium and tracheal muscle following intranasal deliver ofy loaded mini-nucleosomes (Figur e17). id="p-368" id="p-368" id="p-368" id="p-368" id="p-368"

[0368] When injected intranasall they, loaded mini-nucleosomes may be deliver edat a dose greater than le5 genome copies per kg and up to a dose of 1625 copies per kg of body weight (e.g., at about le5, le6, le7, le8, le9, lelO, 1 el 5, 1620, or 1625 copies per kg of body weight, or any range there between). Volume of the material may range from 1- 200 milliliter s (e.g., 1, 5, 10, 20, 30, 40, 50, 100, or 200 milliliter Thes). loaded mini-nucleosomes may also be administered repeatedly. The loaded mini-nucleosomes may also be deliver edorally to access gut, pancreas etc.

Example 7: Route of administration- Intramuscular id="p-369" id="p-369" id="p-369" id="p-369" id="p-369"

[0369] This example demonstrate thats loaded mini-nucleosomes can be deliver edby intra-muscular route to express proteins in the muscle cells. Balb/c mice were anesthetized by IP injection with Ketamine/Xylazine (90-100 mg/kg +10 mg/kg) and several loaded mini - nucleosomes were injected into both leg muscle at 17.5ug doses per leg using an insulin syringe (Total dose 35 micrograms per mice). Mice were sacrificed at various time points and leg muscle were obtained for tissue sections. Constructs that contained core proteins such as polylysine (SEQ ID NO: 393) or mini-nucleosome with other domain combinations (SEQ ID NO: 389) didn’t exhibit GFP fluorescence at the 3-month time point. Surprisingly, in muscle tissue sections obtained from 3-months post injections, we observed sharp green fluorescence in skelet muscleal cells injected with loaded mini-nucleosomes with containing galactose and fucos ebinding domain as shown in SEQ ID NO: 391 (Fig 18 A, B and C). This demonstrate s that some domains have a higher propensity of attachment and internalization into muscle cells and could be utilized for efficie ntgene transf erto muscle cell s.One skilled in the art may contemplate combining such domains with other domains known for muscle tropism. id="p-370" id="p-370" id="p-370" id="p-370" id="p-370"

[0370] To validate muscle specificit ofy expression of genes encoded by the nucleic acid cargo, we utilized dystrophin immunolabeling as an endogenous secondary marker .Regions of sharp green fluorescen (panelce A) encircled by red fluorescence (panel B; merged in panel C) of 148 Dystrophin staining clearly demonstrate thats loaded mini-nucleosomes injected intramuscularly can deliver genes to muscle cells (Fig 18). Native fluorescence of GFP was obtained in the 486- nm channel of Leica SP5 scope. Dystrophin in red is the RFP channel (555-nm). Untransduced muscle cells in figur ealso serve as internal control for differentiation between GFP signal and autofluorescence. id="p-371" id="p-371" id="p-371" id="p-371" id="p-371"

[0371] Provided herein are technique tos deliver loaded mini-nucleosomes into muscle cells by intramuscular mode of deliver Manyy. genetic muscular dystrophies lead to atrophy of the muscle cell s.To deliver functional genes to these muscle cells, intramuscula router provides direct routes of administration. We demonstrated the muscle tropism and ability of loaded-mini- nucleosomes to express genes in the skelet muscleal cells (Figurel8). Expression was observed in muscle cells as early as day 2 afte deliverr y.Provided herein are muscle-tropic domains that could enhance vector uptake and gene expression, howeve ris not limited to it. We also observed that spiral shaped loaded mini-nucleosomes deliver edvia intramuscular route, transduce muscle cells effectively and for longer durations- in this case 3 months (Figur e18) compared to lobular shaped molecule (data not shown). The shape of vector hass not been described before in the contex oft delivering genes to the muscle cell s.One skilled in the art may contemplat utilie, zing other structur esfor increased cell tropism for muscle cell s.Overall, the expression of GFP in dystrophin expressing muscle cells demonstrate thes ability of loaded mini-nucleosomes to rescue diseases like Duchenne muscular dystrophy or other muscular dystrophies. Muscle tropism may also be enhanced by inclusion of other domains described in Table 4. Muscle tropism may also be achieved by intravenous delivery. id="p-372" id="p-372" id="p-372" id="p-372" id="p-372"

[0372] When injected via intramuscular route the, loaded mini-nucleosomes may be deliver edat a dose greater than le5 genome copies per kg and up to a dose of 1625 copies per kg of body weight (e.g., at about le5, le6, le7, le8, le9, lelO, 1615, le20, or 1625 copies per kg body weight, or any range there between). Volume of the material may range from 1- 900 milliliter (e.g.,s 1, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 milliliter Thes). loaded mini-nucleosomes may also be administered repeatedly (e.g., a selected volume and/or number of genome copies can be administered multiple times or divided among two or more does). The loaded mini-nucleosomes may also be administered intravenousl to y access muscle cells. 149 Example 8: Loaded mini-nucleosomes are redosable id="p-373" id="p-373" id="p-373" id="p-373" id="p-373"

[0373] This example demonstrates that mini-nucleosomes can be re-administer withouted any neutralizing effect on the expression of proteins (Figur e19). Balb/c mice were simply restrained using standard restraining techniques and Insuli nsyringe were used to deliver the loaded mini-nucleosomes MN #1 (SEQ ID NO: 390 + F8 plasmid), and MN #2 (SEQ ID NO: 391 + F8 plasmid, SEQ ID NO: 393) via tail vein injection. Each mouse received 20 micrograms 1st dose and 40 micrograms 2nd dose (30 days afte 1rst dose). Serum were collected by cheek bleed techniqu ate day 3 post 1st and 2nd doses. ~150ul blood were collected each time and serum was collected from blood using standar techniques.d F8 Elisa was performed to determine expression levels of F8 in serum in Balb/c mice following intravenous deliver ofy loaded mini - nucleosomes. F8 Elisa was performed according to manufactur’ser (Aviva Systems Biology) instructions. 1:6 serum dilutions were made for all assays. We observed that when deliver eda second time, there was no neutralizing effect in the expression levels, as evidenc edby increase in protein level ofs F8 (Figure 19). id="p-374" id="p-374" id="p-374" id="p-374" id="p-374"

[0374] Provided herein are example ofs mini-nucleosome core proteins and loaded mini - nucleosome that can be delivered repeatedly to boost expression level ofs desired proteins.

Redosabili tyis a very important featur fore any drug that may require repeat administrati on.In gene therapy, current lyone of the most undesirable features of viral vector iss the inability to re- administ erdrug products Viral. vector once injected into the patient leads to formation of neutralizing antibodies. This cause simmunogenicity and inexpressibilit wheny they are administered the second time. We show here that mini-nucleosome, mediated gene delivery solves this problem. The non-immunogenic nature of mini-nucleosom is eengineered in by design: by combining self-peptides or human derived amino acid sequences and enhanced by pegylation. In literature, pegylate proteinsd have been shown to evade the immune system. In this case, in mice, lack of immunogenicity for artificial human derived core proteins, further validat thees case for pegylation. This redosability featur wille allow multiple treatments to patien tswhen needed. In case of diminishing expression level overs time, this redosable featur e will allow repeat treatment to boost the expression to desired levels This. piece of data also shows that in some patien tsthat need multi-organ injections, mini-nucleosom mediatede gene 150 transf erwill be most desirable. One skilled in the art may also contemplate repeat dosing via many other routes of administrat ionsuch as topical oral,, vaginal intr, aperitoneal intr, aocular, intrathecal, intracerebral subcutaneous, etc. or via encapsulation in liposome ors other synthetic materials. id="p-375" id="p-375" id="p-375" id="p-375" id="p-375"

[0375] Repea tdoses may be deliver edat a concentration greater than le5 genome copies per kg and up to a dose of 1625 copies per kg of body weight (e.g., at about le5, le6, le7, le8, le9, lelO, 1615, le20, or 1625 copies per kg body weight, or any range there between). Volume of the material may range from 1- 900 milliliter (e.g.,s 1, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 milliliter Thes). loaded mini-nucleosomes may also be administered repeatedly (e.g., a selected volume and/or number of genome copies can be administered multiple times or divided among two or more does).

Example 9: General techniques id="p-376" id="p-376" id="p-376" id="p-376" id="p-376"

[0376] This example describes genera techl nique fors cloning, deliver ofy min- nucleosomes into cells. Some of the cloning techniques that can be applied to constructing these vector mays include- synthesis of transge neconstructs, TOPO PCR cloning, blunt end cloning, seamless cloning, long fragment cloning, restriction enzyme digestion and ligation but not limited to these technique DNAs. or RNA molecules may express one or more expression marker ssuch as GFP, YEP and Luciferase but not limited to it. DNA or RNA molecules may express one or more therapeut RNAic or proteins but not limited to it. id="p-377" id="p-377" id="p-377" id="p-377" id="p-377"

[0377] Loaded mini-nucleosomes can be tested for their function and characterized in vitro by expressing them in HEK cells or other animal cell lines. Ability of synthesized and/or purified loaded mini-nucleosomes to transduce hematopoieti stemc cells or differentiated peripheral blood mononuclear cells can be assayed by exposing the cells to the loaded mini - nucleosomes in culture. Loaded mini-nucleosomes can also be tested for thei rfunction and ability to form chimeric T cells in vitro by exposure to mini-nucleosomes or via techniques of transfection, or other physical methods for insertions. Loaded mini-nucleosomes can be tested for thei rfunction and characterized in vivo by delivering in mice or any other animal models but not limite tod it. 151 Example 10: Phosphorylated Mini-Nucleosome Core Proteins Deliver Nucleic Acid Payloads to Neurons and Central Nervous System Cells id="p-378" id="p-378" id="p-378" id="p-378" id="p-378"

[0378] The present Example demonstrates that a mini-nucleosome core protein including one or more residues modified by phosphorylation is particularly advantageou at sleast for expression of a nuclei acidc payload (i.e., expression of an expression product encoded by a nucleic acid payload) in certain cells or tissues. For example, a mini-nucleosom coree protein including one or more residues modified by phosphorylation can be particularly advantageo us for expression of a nucleic acid payload in central nervous system cells including neurons and, particularly including spinal cord cells and brain neurons. id="p-379" id="p-379" id="p-379" id="p-379" id="p-379"

[0379] Modified mini-nucleosome core proteins were prepared by phosphorylation of a mini-nucleosome core protein having an amino acid sequenc accore ding to SEQ ID NO: 399. In particular a, threonine residue at position 11 of the mini-nucleosom coree protein was phosphorylated. This residue is positioned in a linker domain (VT) of the mini-nucleosome core protein. However the, present disclosure provides that the presence of the modification, rathe r than the particular position of the modification within the mini-nucleosome core protein, determines the characteristi csof the mini-nucleosome core proteins disclosed herein. Without wishing to be bound by any particular scientif theoric y, the present Example includes the recognition that modifications of mini-nucleosom coree proteins according the present Example can interact with cell surfac ereceptors improving, deliver ofy loaded mini-nucleosomes to certain target cell s.Accordingl y,the present disclosure provides that phosphorylation of any amino acid residue(s) of a mini-nucleosome core protein, in particular a serine, threonine, or tyrosine, would provide equivalent advantages and characteristics as phosphorylation of the particular residu eof the particular mini-nucleosom coree protein modified in the present Example. Unmodified mini-nucleosom coree proteins according to the same amino acid sequenc weree also included in the present example as a control. id="p-380" id="p-380" id="p-380" id="p-380" id="p-380"

[0380] Mini-nucleosome core proteins of the present Example were loaded with a nucleic acid payload that included a gene encoding luciferase. In the present Example, lucifera seis representati ofve protein expression generally in ,that expression of luciferase is indicative of the ability of a mini-nucleosom coree protein modified as disclosed herein and discusse din the present Example to successfully delivery any nucleic acid payload for expression of any 152 expression product .Loaded mini-nucleosomes of the present Example were administered intrathecally to one or more 9-10 week old Balb/c mice (3el0 gc/mouse; lOul volume). id="p-381" id="p-381" id="p-381" id="p-381" id="p-381"

[0381] Result ares shown in Figure 31. On day 11 post dose administrati on,mice were dosed with luciferin at 150 mg/kg (60 mg/mL) via intraperitoneal (IP) injection at 2.5 ml/kg. At ~15 minutes post each lucifer inadministrat ion(± 5%), all animals underwent an IVIS imagin g session. Panel A of Figure 31 shows resul tsusing loaded mini-nucleosomes including an unmodified mini-nucleosom coree protein, while Panel B of Figure 31 shows results using loaded mini-nucleosomes including a phosphorylated mini-nucleosome core protein. All mini- nucleosome core proteins were loaded with a nucleic acid payload including a gene encoding luciferase. Panel B shows that the modified mini-nucleosome core protein, but not the unmodified mini-nucleosom coree protein results in robust expression of the representative nucleic acid payload-encoded expression product (here, luciferase) in certain tissues including centr alnervou systems cells including neurons, and including spinal cord cells and brain neurons. Expression level observeds in the present Example were highly unexpected. Detection of expression by IVIS imaging requires a very high level of expression, and the present data therefor refle ects remarkable efficienc ofy both cellul uptakear and payload expression. Levels of expression dramatically exceed those that would be expected for a non-viral vector.

Example 11: Sulfated Mini-Nucleosome Core Proteins Deliver Nucleic Acid Payloads to Neurons and Central Nervous System Cells id="p-382" id="p-382" id="p-382" id="p-382" id="p-382"

[0382] The present Example demonstrates that a mini-nucleosome core protein including one or more residues modified by sulfati onis particularly advantageou at sleast for expression of a nucleic acid payload (i.e., expression of an expression product encoded by a nucleic acid payload) in certain cells or tissues. For example, a mini-nucleosome core protein including one or more residues modified by sulfati oncan be particularly advantageou fors expression of a nucleic acid payload in centr alnervous system cells including neurons, and particularly including spinal cord cells and brain neurons. id="p-383" id="p-383" id="p-383" id="p-383" id="p-383"

[0383] Modified mini-nucleosome core proteins were prepared by sulfati onof a mini - nucleosome core protein having an amino acid sequenc accore ding to SEQ ID NO: 388. In particular a, tyrosine residu eat position 38 of the mini-nucleosome core protein was sulfated. 153 This residue is positioned in a targeting domain (FYQPL) of the mini-nucleosome core protein.

However, the present disclosure provides that the presence of the modification, rather than the particular position of the modification within the mini-nucleosom coree protein, determines the characteristi csof the mini-nucleosome core proteins disclosed herein. Without wishing to be bound by any particular scientif theory,ic the present Example includes the recognition that modifications of mini-nucleosom coree proteins according the present Example can interact with cell surfac ereceptors, improving delivery of loaded mini-nucleosomes to certain target cells.

Accordingl y,the present disclosure provide thats sulfation of any amino acid residue( s)of a mini-nucleosom coree protein, in particular a serine, threonine, or tyrosine, would provide equivalent advantages and characteristi csas sulfation of the particular residue of the particul ar mini-nucleosom coree protein modified in the present Example. Unmodifie mini-nucleosomed core proteins according to the same amino acid sequenc weree also included in the present example as a control. id="p-384" id="p-384" id="p-384" id="p-384" id="p-384"

[0384] Mini-nucleosome core proteins of the present Example were loaded with a nucleic acid payload that included a gene encoding luciferase. In the present Example, lucifera seis representati ofve protein expression generally in ,that expression of luciferase is indicative of the ability of a mini-nucleosom coree protein modified as disclosed herein and discusse din the present Example to successfully delivery any nucleic acid payload for expression of any expression product .Loaded mini-nucleosomes of the present Example were administered intrathecally to one or more 9-10 week old Balb/c mice (3el0 gc/mouse; lOul volume). id="p-385" id="p-385" id="p-385" id="p-385" id="p-385"

[0385] Result ares shown in Figures 32 and 33. On day 11 post dose administration, mice were dosed with lucifer inat 150 mg/kg (60 mg/mL) via intraperitoneal (IP) injection at 2.5 ml/kg. At ~15 minutes post each luciferin administrat ion(± 5%), all animals underwent an IVIS imaging session. Panel A of Figure 32 shows results using loaded mini-nucleosomes including an unmodified mini-nucleosome core protein, while Panel B of Figure 32 shows resul tsusing loaded mini-nucleosomes including a sulfate mini-nucleosomed core protein. All mini - nucleosome core proteins were loaded with a nucleic acid payload including a gene encoding luciferase. Panel B of Figure 32 shows that the modified mini-nucleosome core protein, but not the unmodified mini-nucleosome core protein results in robust expression of the representative nucleic acid payload-encoded expression product (here, luciferase) in certain tissues including 154 centr alnervou systems cells including neurons, and including spinal cord cells and brain neurons. Figure 33 includes brain tissue section ofs a mouse administered a sulfate minid - nucleosome core protein loaded with the luciferase-encodi payload,ng which mouse showed a high degree of expression in certain tissues including centr alnervou systems cells including neurons, and including spinal cord cells and brain neuron (Panels A). Panels B, C, and D respectively show luciferas e,anti-NeuN antibody, and D-D API stains, with an overla yof these stains shown in Panel E. Images of Figure 33 demonstrate robust expression of the representati nucleicve acid payload-encoded expression product (here, luciferase) in brain cell s, particularly including brain neurons, when the mini-nucleosome is sulfate butd not when the mini-nucleosom is eunmodified. Expression level obsers ved in the present Example were highly unexpected. Detection of expression by IVIS imaging requires a very high level of expression, and the present data therefor refe lects remarkable efficienc ofy both cellul uptakear and payload expression. Levels of expression dramaticall exceedy those that would be expected for a non- viral vector. Moreover ,it was unexpected that sulfati onwould increase expression in brain cells, particularly including brain neurons.

Example 12: Acetylated Mini-Nucleosome Core Proteins Deliver Nucleic Acid Payloads to Neurons and Central Nervous System Cells id="p-386" id="p-386" id="p-386" id="p-386" id="p-386"

[0386] The present example demonstrate thats a mini-nucleosome core protein including one or more residues modified by acetylation is particularly advantageous at least for expression of a nucleic acid payload (i.e., expression of an expression product encoded by a nuclei acidc payload) in certain cells or tissues. For example a mini-nucleosom coree protein including one or more residues modified by acetylati oncan be particularly advantageou fors expression of a nucleic acid payload in retinal cells, in particular photoreceptors id="p-387" id="p-387" id="p-387" id="p-387" id="p-387"

[0387] Modified mini-nucleosome core proteins were prepared by acetylation of a mini - nucleosome core protein having an amino acid sequenc accore ding to SEQ ID NO: 401. In particular a, lysine residue at position 10 of the mini-nucleosom coree protein was acetylated.

This residue is positioned in a targeting domain (KKRPKP) of the mini-nucleosome core protein.

However, the present disclosure provides that the presence of the modification, rather than the particular position of the modification within the mini-nucleosom coree protein, determines the 155 characteristi csof the mini-nucleosome core proteins disclosed herein. Without wishing to be bound by any particular scientif theory,ic the present Example includes the recognition that modifications of mini-nucleosom coree proteins according the present Example can interact with cell surfac ereceptors, improving delivery of loaded mini-nucleosomes to certain target cells.

Accordingl y,the present disclosure provide thats acetylation of any amino acid residue(s) of a mini-nucleosom coree protein, in particular a lysine, would provide equivalent advantages and characteristi csas acetylation of the particular residu eof the particular mini-nucleosome core protein modified in the present Example. Unmodified mini-nucleosome core proteins according to the same amino acid sequenc weree also included in the present example as a control. id="p-388" id="p-388" id="p-388" id="p-388" id="p-388"

[0388] Mini-nucleosome core proteins of the present Example were loaded with a nucleic acid payload that included a gene encoding GFP. In the present Example, GFP is representative of protein expression generally in ,that expression of GFP is indicative of the ability of a mini - nucleosome core protein modified as disclosed herein and discusse din the present Example to successfull delivery anyy nucleic acid payload for expression of any expression product.

Loaded mini-nucleosomes of the present Example were administered intravitreal toly one or more mice. For intravitreal injections, mice were anesthetized by IP injection with Ketamine/Xylazine (90-100 mg/kg +10 mg/kg), a target pupil was dilated under a microscope using topical Tropicamide (1%), and an incision/insertion was made on the scler a~1 mm below the limbus using a 30-gauge needle. Ipl of loaded mini-nucleosome was injected into the vitreous cavity using 32 gauge blunt needle passing through the incision made by the 25-gauge needle. id="p-389" id="p-389" id="p-389" id="p-389" id="p-389"

[0389] Result ares shown in Figure 34. Eyes were enucleat ated 4 weeks post injection and place din 4%PFA for fixation. Lenses were removed from the eyes by dissection and the retinal wholemount was dissected from eyecup. Retinal wholemounts were hydrated with IxPBS, blocked using 2% BSA and staine withd GFP antibodi escoupled to Alexa-flour 555 at room temperature for 1 hr and washed with IxPBS 4 times, 5 minutes each. Images were obtained using Leica SP5 confocal microscope. id="p-390" id="p-390" id="p-390" id="p-390" id="p-390"

[0390] Panels A-C of Figure 34 show results using loaded mini-nucleosomes including an unmodified mini-nucleosome core protein, while Panel D-F of Figure 34 shows results using loaded mini-nucleosomes including an acetylated mini-nucleosome core protein. All mini 156 nucleosome core proteins were loaded with a nucleic acid payload including a gene encoding GFP. Images demonstrate robust expression of the representati nucleicve acid payload-encoded expression product (here, GFP) in retinal cells, in particular photoreceptor whens, the mini - nucleosome is acetylated but not when the mini-nucleosome is unmodified. Expression levels observed in the present Example were highly unexpected. Detection of expression by IVIS imaging requires a very high level of expression, and the present data therefor refle ects remarkable efficiency of both cellul uptakear and payload expression. Levels of expression dramatically exceed those that would be expected for a non-viral vector. Moreover ,it was unexpecte thatd acetylation would increase expression in retinal cell s,in particul ar photoreceptors.

Example 13: Phosphorylated Mini-Nucleosome Core Proteins Deliver Nucleic Acid Payloads to Neurons and Central Nervous System Cells id="p-391" id="p-391" id="p-391" id="p-391" id="p-391"

[0391] The present Example demonstrates that a mini-nucleosome core protein including one or more residues modified by mannosylation is particularly advantageous at least for expression of a nuclei acidc payload (i.e., expression of an expression product encoded by a nucleic acid payload) in certain cells or tissues. For example, a mini-nucleosom coree protein including one or more residues modified by mannosylati oncan be particularly advantageou fors expression of a nuclei acidc payload in retinal cells, in particular photoreceptors. id="p-392" id="p-392" id="p-392" id="p-392" id="p-392"

[0392] Modified mini-nucleosome core proteins were prepared by mannosylation of a mini-nucleosom coree protein having an amino acid sequenc accore ding to SEQ ID NO: 447. In particular a, serine residue at position 9 of the mini-nucleosom coree protein was mannosylated.

This residue is positioned in a linker domain (GGS) of the mini-nucleosome core protein.

Accordingl y,the present disclosure provide thats mannosylation of any amino acid residue(s) of 157 a mini-nucleosome core protein, in particular a serine, would provide equivalent advantages and characteristi csas mannosylation of the particular residue of the particular mini-nucleosom coree protein modified in the present Example. Unmodified mini-nucleosome core proteins according to the same amino acid sequenc weree also included in the present example as a control. id="p-393" id="p-393" id="p-393" id="p-393" id="p-393"

[0393] Mini-nucleosome core proteins of the present Example were loaded with a nucleic acid payload that included a gene encoding GFP. In the present Example, GFP is representative of protein expression generally in ,that expression of GFP is indicative of the ability of a mini - nucleosome core protein modified as disclosed herein and discusse din the present Example to successfull delivery anyy nucleic acid payload for expression of any expression product.

Loaded mini-nucleosomes of the present Example were administered subretinally to one or more mice. For subretina injectl ions, mice were anesthetized by IP injection with Ketamine/Xylazine (90-100 mg/kg +10 mg/kg), a target pupil was dilated under a microscope using topical Tropicamide (1%), and an incision/insertion was made on the sclera ~1 mm below the limbus using a 30-gaug eneedle Ipl. of loaded mini-nucleosome was injected into the vitreous cavity using 32 gauge blunt needle passing through the incision made by the 25-gauge needle behind the retina. A micropump controlled injector was utilized for this procedure. id="p-394" id="p-394" id="p-394" id="p-394" id="p-394"

[0394] Result ares shown in Figure 35. Eyes were enucleat ated 2 weeks post injection and place din 4%PFA for fixation. Lenses were removed from the eyes by dissection and the retinal wholemount was dissected from eyecup. Retinal wholemounts were hydrated with IxPBS, blocked using 2% BSA and staine withd GFP antibodi escoupled to Alexa-flour 555 at room temperature for 1 hour and washed with IxPBS 4 times 5, minutes each. Images were obtained using Leica SP5 confocal microscope. id="p-395" id="p-395" id="p-395" id="p-395" id="p-395"

[0395] Figure 35, panels A-D, show a representati retive nal wholemount from a mouse administered loaded mini-nucleosomes including mannosylated mini-nucleosom coree protein.

Image sdemonstrate robust expression of the representati nucleive acidc payload-encoded expression product (here, GFP) in retinal cells, in particular photoreceptor whens, the mini - nucleosome is mannosylated. An unmodified control loaded with the same nuclei acidc payload was not robustly expressed in retinal cells or photoreceptors. Expression level observeds in the present Example were highly unexpected. Detection of expression by IVIS imaging requires a very high level of expression, and the present data therefor refe lects remarkable efficiency of 158 both cellul uptakear and payload expression. Levels of expression dramatically exceed those that would be expected for a non-viral vector. 159 CERTAIN SEQUENCES: SEQIDNO: 394 KKRHRK-[LINKER]-LRE-[LINKER] KRHRKLRRRRRLKRHRKKRHRK-[LINKER]- ERE-[LINKER]-K (where [LINKER] could be any amino acid sequenc describede in Table 12 but not limited to it) SEQIDNO: 389 KKKRHRKRKRKRKRRRRKKK-[LINKER]-ASSLNIAK-[LINKER]-RRRR (where [LINKER] could be any amino acid sequenc describede in Table 12 but not limited to it) SEQIDNO: 390 KKKRK-[LINKER]-NGR-[LINKER] -KRKRKKRHRKKKKRRRRKRHRK-[LINKER] - NGR-[LINKER]-KKK (where [LINKER] could be any amino acid sequenc describede in Table 12 but not limited to it) SEQIDNO: 391 KKKRHRKKKKK-[LINKER] -RGD-[LINKER] -KKKK-[LINKER] -NTQIH-[LINKER] - RRRRR-[LINKER] -TPH-[LINKER] -KK (where [LINKER] could be any amino acid sequenc describede in Table 12 but not limited to it) SEQIDNO: 392 KKKRK-[LINKER]-KTKKK-[LINKER]-AK-[LINKER]-KALKKK-[LINKER]- KKGKKKKRRRRKAAPKK (where [LINKER] could be any amino acid sequenc describede in Table 12 but not limited to it) SEQIDNO: 393 CKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK 160 SEQIDNO: 460 CBA-F8 plasmid TCGCGCGTTTCGGTGATCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCC CCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATG GGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGG CGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCC GAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGC GCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGC CGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGG GCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGT TTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGG GGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGC GGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCG CTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGG GGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCA GGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGT TGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGC TCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCC GCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGC GGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAG GGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCC GCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGA AATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCA GCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGG GGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATG CCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCAT TTTGGCAAAACCGGTCTCGAAGGCCTGCAGGCGGCCGCCGCCACCGCCACCATGCA AATAGCACTCTTCGCTTGCTTCTTTCTGAGCCTTTTCAATTTCTGCTCTAGTGCCATCA GAAGATACTACCTTGGTGCAGTGGAATTGTCCTGGAACTATATTCAGAGTGATCTGC TCAGTGTGCTGCATACAGACTCAAGATTTCTTCCTAGAATGTCAACATCTTTTCCATT 161 CAACACCTCCATCATGTATAAAAAGACTGTGTTTGTAGAGTACAAGGACCAGCTTTT CAACATTGCCAAGCCCAGGCCACCCTGGATGGGTTTGCTAGGTCCTACCATTTGGAC TGAGGTTCATGACACAGTGGTCATTACACTTAAAAACATGGCTTCTCATCCTGTCAG TCTTCATGCTGTTGGTGTGTCCTACTGGAAAGCTTCTGAGGGAGATGAATATGAAGA TCAGACAAGCCAAATGGAGAAGGAAGATGATAAAGTTTTCCCTGGTGAAAGTCATA CTTATGTTTGGCAAGTCCTGAAAGAGAATGGTCCAATGGCCTCTGACCCTCCATGTC TCACTTACTCATATATGTCTCATGTGGATCTGGTGAAAGATTTGAATTCAGGCCTCAT TGGAGCTCTGCTAGTATGTAAAGAAGGCAGTCTCTCCAAAGAAAGAACACAGATGT TGTACCAATTTGTACTGCTTTTTGCTGTATTTGATGAAGGGAAGAGCTGGCACTCAG AAACAAACGACTCTTATACACAGTCTATGGATTCTGCATCTGCTAGAGACTGGCCTA AAATGCACACAGTCAATGGCTATGTAAACAGGTCTCTTCCAGGTCTGATTGGATGCC ATAGGAAATCAGTCTACTGGCACGTGATTGGAATGGGCACCACTCCTGAAATACACT CAATATTCCTCGAAGGTCACACATTTTTTGTGAGGAACCACCGTCAAGCTTCATTGG AGATATCACCAATAACTTTCCTTACTGCTCAAACACTCTTGATAGATCTTGGGCAGTT CCTACTATTTTGTCATATCTCTTCCCATAAACATGATGGCATGGAAGCTTATGTCAAA GTAGATAGCTGCCCTGAGGAATCCCAATGGCAAAAGAAAAATAATAATGAGGAAAT GGAAGATTATGATGATGATCTTTATTCAGAAATGGATATGTTCACATTGGATTATGA CAGCTCTCCTTTTATCCAAATTCGCTCGGTTGCTAAAAAGTACCCTAAAACTTGGAT ACATTATATTTCTGCTGAGGAGGAAGACTGGGACTATGCACCTTCAGTTCCTACCTC GGATAATGGAAGTTATAAAAGCCAGTATCTGAGCAATGGTCCTCATCGGATTGGTA GGAAATATAAAAAAGTCAGATTTATAGCATACACAGATGAAACCTTTAAGACTCGT GAAACTATTCAGCATGAATCAGGACTCTTGGGACCTTTACTTTATGGAGAAGTTGGA GACACACTGTTGATTATTTTTAAGAATCAAGCAAGCCGACCATATAACATTTACCCT CATGGAATCACTGATGTCAGTCCTCTACATGCAAGGAGATTGCCAAGAGGTATAAA GCACGTGAAGGATTTGCCAATTCATCCAGGAGAGATATTCAAGTACAAGTGGACAG TTACAGTAGAAGATGGACCAACTAAATCAGATCCACGGTGCCTGACCCGCTATTATT CAAGTTTCATTAACCCTGAGAGAGATCTAGCTTCAGGACTGATTGGCCCTCTTCTCA TCTGCTACAAAGAATCTGTAGATCAAAGGGGAAACCAGATGATGTCAGACAAAAGA AATGTCATCCTGTTTTCTATATTTGATGAGAACCAAAGCTGGTACATCACAGAGAAC ATGCAACGCTTCCTCCCCAATGCAGCTAAAACACAGCCCCAGGACCCTGGGTTCCAG 162 GCCTCCAACATCATGCACAGCATCAATGGCTATGTTTTTGATAGCTTGGAGTTGACA GTTTGTTTGCATGAGGTGGCATACTGGCACATTCTCAGTGTTGGAGCACAGACAGAC TTCTTATCTATCTTCTTCTCTGGATATACTTTCAAACACAAAATGGTCTATGAAGATA CACTTACCCTGTTCCCATTCTCAGGAGAAACTGTCTTTATGTCGATGGAAAACCCAG GTCTATGGGTCTTGGGGTGTCATAATTCAGACTTTCGGAAGAGAGGTATGACAGCAT TGCTGAAAGTTTCTAGTTGTGACAAGAGCACTAGTGATTATTATGAAGAAATATATG AAGATATTCCAACACAGTTGGTGAATGAGAACAATGTCATTGATCCCAGAAGCTTCT TCCAGAATACAAATCATCCTAATACTAGGAAAAAGAAATTCAAAGATTCCACAATT CCAAAAAATGATATGGAGAAGATTGAGCCTCAGTTTGAAGAGATAGCAGAGATGCT TAAAGTACAGAGTGTCTCAGTTAGTGACATGTTGATGCTCTTGGGACAGAGTCATCC TACTCCACATGGCTTATTTTTATCAGATGGCCAAGAAGCCATCTATGAGGCTATTCA TGATGATCATTCACCAAATGCAATAGACAGCAATGAAGGCCCATCTAAAGTGACCC AACTCAGGCCAGAATCCCATCACAGTGAGAAAATAGTATTTACTCCTCAGCCCGGCC TCCAGTTAAGATCCAATAAAAGTTTGGAGACAACTATAGAAGTAAAGTGGAAGAAA CTTGGTTTGCAAGTTTCTAGTTTGCCAAGTAATCTAATGACTACAACAATTCTGTCAG ACAATTTGAAAGCAACTTTTGAAAAGACAGATTCTTCAGGATTTCCAGATATGCCAG TTCACTCTAGTAGTAAATTAAGTACTACTGCATTTGGTAAGAAAGCATATTCCCTTGT TGGGTCTCATGTACCTTTAAACGTGAGTGAAGAAAATAGTGATTCCAACATATTGGA TTCAACTTTAATGTATAGTCAAGAAAGTTTACCAAGAGATAATATATTATCAATGGA GAATGATAGATTACTCAGAGAGAAGAGGTTTCATGGAATTGCTTTATTGACCAAAG ATAATACTTTATTCAAAGACAATGTCTCCTTAATGAAAACAAACAAAACATATAATC ATTCAACAACTAATGAAAAACTACACACTGAGAGCCCAACATCAATTGAGAATAGT ACAACAGACTTGCAAGATGCCATATTAAAGGTCAATAGTGAGATTCAAGAAGTAAC AGCTTTGATTCATGATGGAACACTTTTAGGCAAAAATTCTACATATTTGAGACTAAA CCATATGCTAAATAGAACTACCTCAACAAAAAATAAAGACATATTTCATAGAAAAG ATGAAGATCCTATTCCACAAGATGAAGAGAATACAATCATGCCATTTTCCAAGATGT TGTTCTTGTCAGAATCTTCAAATTGGTTTAAAAAGACCAATGGAAATAATTCCTTGA ACTCTGAGCAAGAACATAGTCCAAAGCAATTAGTATATTTAATGTTTAAAAAATATG TAAAAAATCAAAGTTTCTTGTCAGAGAAAAATAAAGTCACAGTAGAACAGGATGGA TTTACAAAGAACATAGGACTTAAAGACATGGCTTTTCCACATAATATGAGCATATTT 163 CTTACCACTTTGTCTAACGTACATGAAAATGGTAGGCACAATCAAGAAAAAAATATT CAGGAAGAGATAGAGAAGGAAGCACTAATTGAAGAGAAAGTAGTTTTGCCCCAGGT GCACGAAGCAACTGGCTCTAAGAATTTCTTGAAAGACATATTGATACTAGGCACTAG GCAAAATATAAGTTTATATGAAGTACATGTACCAGTACTTCAAAACATCACATCAAT AAACAATTCAACAAATACAGTACAGATTCACATGGAGCATTTCTTTAAAAGAAGGA AGGACAAGGAAACAAATTCAGAAGGCTTGGTAAATAAAACCAGAGAAATGGTAAA AAACTATCCAAGCCAGAAGAATATTACTACTCAACGTAGTAAACGGGCTTTGGGAC AATTCAGACTGTCAACTCAATGGCTTAAAACCATAAACTGTTCAACACAGTGTATCA TTAAACAGATAGACCACAGCAAGGAAATGAAAAAGTTCATTACTAAATCTTCCTTAT CAGATTCTTCTGTGATTAAAAGCACCACTCAGACAAATAGTTCTGACTCACACATTG TAAAAACATCAGCATTTCCACCAATAGATCTCAAAAGGAGTCCATTCCAAAACAAA TTTTCTCATGTTCAAGCATCATCCTACATTTATGACTTTAAGACAAAAAGTTCAAGA ATTCAAGAAAGCAATAATTTCTTAAAAGAAACCAAAATAAATAACCCTTCTTTAGCC ATTCTACCATGGAATATGTTCATAGATCAAGGAAAATTTACCTCCCCAGGGAAAAGT AACACAAACTCAGTCACATATAAGAAACGTGAGAACATTATTTTCTTGAAACCAACT TTGCCTGAAGAATCTGGCAAAATTGAATTGCTTCCTCAAGTTTCCATTCAAGAGGAA GAAATTTTACCTACAGAAACTAGCCATGGATCTCCTGGACACTTGAATCTCATGAAA GAGGTCTTTCTTCAGAAAATACAGGGGCCTACTAAATGGAATAAAGCAAAGAGGCA TGGAGAAAGTATAAAAGGTAAAACAGAGAGCTCTAAAAATACTCGCTCAAAACTGC TAAATCATCATGCTTGGGATTATCATTATGCTGCACAGATACCAAAAGATATGTGGA AATCCAAAGAGAAGTCACCAGAAATTATATCCATTAAGCAAGAGGACACCATTTTG TCTCTGAGGCCTCATGGAAACAGTCATTCAATAGGGGCAAATGAGAAACAAAATTG GCCTCAAAGAGAAACCACTTGGGTAAAGCAAGGCCAAACTCAAAGGACATGCTCTC AAATCCCACCAGTGTTGAAACGACATCAAAGGGAACTTAGTGCTTTTCAATCAGAAC AAGAAGCAACTGACTATGATGATGCCATCACCATTGAAACAATCGAGGATTTTGAC ATTTACAGTGAGGACATAAAGCAAGGTCCCCGCAGCTTTCAACAGAAAACAAGGCA CTATTTTATTGCAGCTGTGGAACGACTCTGGGACTATGGGATGAGTACATCTCATGT TCTACGAAATAGGTATCAAAGTGACAATGTACCTCAGTTCAAGAAAGTAGTTTTCCA GGAATTTACTGATGGCTCCTTTAGTCAGCCCTTATATCGTGGAGAATTAAATGAACA CCTGGGGTTGTTGGGCCCATATATAAGAGCAGAAGTTGAAGACAACATTATGGTAA 164 CTTTCAAAAACCAGGCCTCCCGTCCCTACTCCTTCTATTCTAGCCTCATTTCTTATAA AGAAGATCAGAGAGGAGAAGAACCTAGAAGAAACTTTGTCAAGCCTAATGAAACC AAAATTTATTTTTGGAAAGTACAACATCATATGGCACCCACAGAAGATGAGTTTGAC TGCAAGGCCTGGGCTTATTTCTCTGATGTTGATCTTGAAAGAGATATGCACTCGGGA TTAATTGGACCCCTTCTGATTTGCCACGCGAACACACTGAATCCTGCTCATGGGAGA CAAGTGTCAGTACAGGAATTTGCTCTGCTTTTCACTATCTTTGATGAGACCAAGAGC TGGTACTTCACTGAAAACGTGAAAAGGAACTGCAAGACACCCTGCAATTTCCAGAT GGAAGACCCCACTTTGAAAGAGAATTATCGCTTCCATGCAATCAATGGTTATGTAAT GGATACCCTACCAGGCTTAGTAATGGCTCAAGATCAAAGGATTCGATGGTATCTTCT CAGCATGGGCAACAATGAGAACATCCAATCTATTCATTTCAGTGGACATGTTTTCAC TGTACGGAAAAAAGAGGAGTATAAAATGGCAGTGTACAACCTCTACCCAGGTGTTT TTGAGACTCTGGAAATGATACCATCCAGAGCTGGAATATGGCGAGTAGAATGCCTT ATTGGCGAGCACTTACAGGCTGGGATGAGCACTCTTTTTCTGGTGTACAGCAAGCAG TGTCAGATTCCTCTTGGAATGGCTTCTGGAAGCATCCGTGATTTCCAGATTACAGCTT CAGGACATTATGGACAGTGGGCCCCAAACCTGGCAAGACTTCATTATTCCGGATCAA TCAATGCCTGGAGTACCAAGGAGCCCTTTTCTTGGATCAAGGTAGATCTGTTGGCAC CAATGATTGTTCATGGCATCAAGACTCAGGGTGCTCGTCAGAAATTTTCCAGCCTTT ATATCTCTCAATTTATCATCATGTATAGCCTGGATGGGAAGAAGTGGCTGAGTTATC AAGGAAATTCCACTGGAACCTTAATGGTTTTCTTTGGCAATGTGGACTCATCTGGGA TTAAGCATAATAGTTTTAATCCTCCAATTATTGCTCGATATATCCGTTTGCACCCCAC TCATTCTAGCATCCGTAGTACTCTTCGCATGGAGTTGATGGGCTGTGATTTAAACAG TTGCAGCATACCATTGGGAATGGAAAGTAAAGTAATATCAGATACACAAATCACTG CCTCATCCTACTTCACCAACATGTTTGCTACTTGGTCTCCTTCACAAGCTCGACTTCA CCTCCAGGGAAGGACTAATGCCTGGCGACCTCAGGTGAATGATCCAAAACAATGGT TGCAAGTGGACTTACAAAAGACAATGAAAGTCACTGGAATAATAACCCAGGGAGTG AAATCTCTCTTTACCAGCATGTTTGTGAAAGAGTTCCTTATTTCCAGCAGTCAAGATG GCCATCACTGGACTCAAATTTTATACAATGGCAAGGTAAAGGTTTTTCAGGGGAATC AGGACTCATCCACACCTATGATGAATTCTCTAGACCCACCATTACTCACTCGCTATC TTCGAATTCACCCCCAGATCTGGGAGCACCAAATTGCTCTGAGGCTTGAGATTCTAG GATGTGAGGCCCAGCAGCAATACTGACCATGGCCCAACTTGTTTATTGCAGCTTATA 165 ATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCAC TGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTC GTTAACTCGAGGGATCCATCGATGTCGACTGCAGAGGCCTGCATGCAAGCTTGGTGT AATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAA CATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAAC TCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCC AGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCG GTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCC GCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCC CCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG TCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG AACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA GCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATT ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAG CCCAATCTGAATAATGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGA GCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAA AAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCA AGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAAT TTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGA ATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCA GCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGA 166 TTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAG GAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCT GAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTG AGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCAT AAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT ACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAGCGATA GATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATC AGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATGGCTCAT AACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATA TTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACGGGCCAGAGCTGCA SEQIDNO: 395 CBA-GFP plasmid TCGCGCGTTTCGGTGATGACGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCC ATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGC AGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGC GCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAA GCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGC TCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAG GTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGA CGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCT TTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCG CCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGG TGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGG GGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCC TCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTG GCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGG GGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGG 167 AGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATC GTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTG GGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGC AGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCT CCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGG GCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACC ATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCT GTCTCATCATTTTGGCAAAACCGGTCTCGAAGGCCTGCAGGCGGCCGCCGCCACCGC CACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCG AGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGC GATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCC GTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGC TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTAC GTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGA GGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACT TCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAC AACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGAT CCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACA CCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGT CCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAATCCATGGC CCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATT TCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAA TGTATCTTATCATGTCTGGATCTCGTTAACTCGAGGGATCCATCGATGTCGACTGCA GAGGCCTGCATGCAAGCTTGGTGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT GTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCC TGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCT TTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGG GAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCG CTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTT 168 ATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAA AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCC CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCC GACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTG GGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTAT CGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGT AACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTC AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCAC GTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAA ATTAAAAATGAAGTTTTAAATCAAGCCCAATCTGAATAATGTTACAACCAATTAACC AATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCA GGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCA CCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGT CCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAG AAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCT TTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCA ACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTG TTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAG CGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTT TTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATG CTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATC TGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATC GGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGC CCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGACGT TTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGT 169 TTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAG ACACGGGCCAGAGCTGCA SEQIDNO: 396 CBA-Luciferase plasmid TCGCGCGTTTCGGTGATGACGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCC ATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGC AGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGG CGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGC GCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAA GCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGC TCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAG GTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGA CGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCT TTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCG CCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGG TGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGG GGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCC TCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTG GCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGG GGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGG AGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATC GTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTG GGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGC AGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCT CCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGG GCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACC ATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCT GTCTCATCATTTTGGCAAAACCGGTCTCGAAGGCCTGCAGGCGGCCGCCGCCACCGC 170 CACCATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGG AAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTT CCTGGAACAATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAG TACTTCGAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACA AATCACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTG GGCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACGT GAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAAAAG GGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAAATTAT TATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGTTCGTCAC ATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCCTTCGATAGG GACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTCTGCCTAAAGGT GTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGCCAGAGATCCTATT TTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTTGTTCCATTCCATCACG GTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGGATTTCGAGTCGTCTTAAT GTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTTCAGGATTACAAGATTCAAAG TGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCGCCAAAAGCACTCTGATTGACAA ATACGATTTATCTAATTTACACGAAATTGCTTCTGGTGGCGCTCCCCTCTCTAAGGAA GTCGGGGAAGCGGTTGCCAAGAGGTTCCATCTGCCAGGTATCAGGCAAGGATATGG GCTCACTGAGACTACATCAGCTATTCTGATTACACCCGAGGGGGATGATAAACCGG GCGCGGTCGGTAAAGTTGTTCCATTTTTTGAAGCGAAGGTTGTGGATCTGGATACCG GGAAAACGCTGGGCGTTAATCAAAGAGGCGAACTGTGTGTGAGAGGTCCTATGATT ATGTCCGGTTATGTAAACAATCCGGAAGCGACCAACGCCTTGATTGACAAGGATGG ATGGCTACATTCTGGAGACATAGCTTACTGGGACGAAGACGAACACTTCTTCATCGT TGACCGCCTGAAGTCTCTGATTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATT GGAATCCATCTTGCTCCAACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCC CGACGATGACGCCGGTGAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGAC GATGACGGAAAAAGAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAA AGTTGCGCGGAGGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAA CTCGACGCAAGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGA TCGCCGTGTAATCCATGGCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA 171 AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT GGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCGTTAACTCGAGGGA TCCATCGATGTCGACTGCAGAGGCCTGCATGCAAGCTTGGTGTAATCATGGTCATAG CTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGC GTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATG AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTC GCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTC AAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTT TTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTA GGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTG CGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCC ACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTA CAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTA TCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCG GCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGC GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTC AGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAAGCCCAATCTGAATA ATGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAA CTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGT AATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCG GTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAA AATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATG GCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTC ATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAG 172 ACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACC GGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTT CTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCAT CAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCA GAAACAACTCTGGCGCATCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATT GCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAAT TTAATCGCGGCCTCGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACT GTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATG TAACATCAGAGATTTTGAGACACGGGCCAGAGCTGCA 173 INCORPORATION BY REFERENCE The publications and patents reference ind this application have been incorporated in their entirety.

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Polyethylenim improvesine the transfect ionefficiency of primary cultur esof post-mitotic rat fet alhypothalamic neurons. JNeurosci Methods 2003;127(2):. 179-92.

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Claims

1. An engineered polypeptide comprising a. nucleic acid binding domain and a nucleic acid release domain, wherein one or more amino acids of the engineered polypeptide is a. modified amino acid, optionally wherein the modification comprises at least one of: phosphorylation, 6) (ii) sulfation; glycosylation; (iii) (iv) prenylation; methylation; (v) (vi) sialylation; lipidation and/or lipoylation; (vii) a x z־C 11 ؛ •ג s !ג ft /xt^ ■ tyln) dLLiyid U Ui 1י (ix) hydroxylation; (x) palmitoylation, (xi) mannosy lati on; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylation; and/or (xv) any combination thereof

2. The engineered polypeptide of claim 1, wherein the engineered polypeptide comprises a. targeting domain.

3. An engineered polypeptide comprising a nucleic acid binding domain and a targeting domain, wherein one or more amino acids of the engineered polypeptide is a modified, amino acid, optionally wherein the modification comprises at least one of. (i) phosphorylation; (ii) sulfation; 184 WO 2021/211467 PCT/US2021/026917 (iii) glycosylation; (iv) prenylation; (v) methylation; (vi) sialylation; (vii) lipidation and/or lipoylation; (viii) acetylation; (ix) hydroxylation; (x) palmitoylation; (xi) mannosylation; (xii) myri stoylation; (xiii) fucosylation; (xiv) pegylation; and/or (xv) any combination thereof; wherein engineered polypeptide optionally further comprises a nucleic acid release domain.

4. The engineered polypepti de of any one of claims 1-3, wherein each of two or more amino acids of the engineered polypeptide is a modified amino acid.

5. The engineered polypeptide of any one of claims 1-4, wherein at least one of the modified amino acids comprises a modification chain comprising two or more modifications selected from: (i) phosphorylation; (ii) sulfation; (iii) glycosylation; (iv) prenylation, (v) methylation; (vi) sialylation; (vii) Lipidation and/or Lipoylation, (viii) acetylation; (ix) hydroxylation; 185 WO 2021/211467 PCT/US2021/026917 (x) paimitoylation; (xi) mannosylation; (xi i) my ri stoy 1 ati on, (xiii) fucosylation; (xiv) pegylation; and (xv) any combination thereof.

6. The engineered polypeptide of any one of claims 1-5, wherein the modification increases the stability, half-life, and/or bioavailability of the engineered polypeptide.

7. The engineered polypeptide of any one of claims 1-6, wherein the modification increases the affinity and/or avidity of the engineered polypeptide with a binding partner, optionally wherein the binding partner is a receptor, cell, or cell membrane.

8. The engineered polypeptide of any one of claims 1-7, wherein the modification increases the affinity or avidity of the engineered polypeptide with a nucleic acid.

9. The engineered polypeptide of any one of claims 1-8, wherein the modification decreases precipitation and/or aggregation of the engineered polypeptide.

10. The engineered polypeptide of any one of claims 1-9, wherein the nucleic acid binding domain is derived from a histone polypeptide sequence.

11. The engineered polypeptide of any one of claims 1-10, wherein the nucleic acid binding domain is or comprises the amino acid sequence KRHRK.

12. The engineered polypeptide of any one of claims 1-11, wherein the nucleic acid binding domain is or comprises an amino acid sequence that, comprises KRHRK, RRRRR, RRLARR, KKAKAAAKPKK, KKDGKKRKR, KKKLK, KKRIRK, RKKSK, KKPKK, or a combination thereof. 186 WO 2021/211467 PCT/US2021/026917

13. The engineered polypeptide of any one of claims 1-12, wherein the nucleic acid binding domain is a. modified nucleic acid binding domain in that the nucleic acid binding domain comprises one or more modified amino acids.

14. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a targeting domain having the sequence of any one of SEQ ID NOs: 397-422, wherein the targeting domain is phosphorylated.

15. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a. targeting domain having the sequence of any one of SEQ ID NOs: 423-428, wherein the targeting domain is sulfated

16., The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a. targeting domain having the sequence of any one of SEQ ID NOs: 429-434, wherein the targeting domain is glycosylated

17. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a targeting domain having the sequence of any one of SEQ ID NOs: 435-440, wherein the targeting domain is prenylated.

18. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a targeting domain having the sequence of any one of SEQ ID NOs: 441-446, wherein the targeting domain is methylated.

19. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a targeting domain having the sequence of any one of SEQ ID NOs: 447-459, wherein the targeting domain is sialylated. 187 WO 2021/211467 PCT/US2021/026917

20. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a cell attachment targeting domain, a beta galactose binding domain, a fucose binding domain, a heparin binding domain, a. sialic acid binding domain, a glycoprotein binding domain, a carbohydrate binding domain, a lysophosphatidic acid, binding domain, a cAMP binding domain, a hyaluronan binding domain, a. chondroitin sulfate binding domain, an integrin binding domain, a nucieolin binding domain, a collagen binding domain, a clathrin binding domain, a Fc receptor binding domain, an actin binding domain, an endocytosis motif, a nuclear localization signal, or a combination thereof.

21. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a. cell attachment targeting domain.

22. The engineered polypeptide of claim 21, wherein the cell attachment targeting domain is or comprises an amino acid sequence that comprises WGREERQ, NTQIH, WNNKTPH, TPH, VNRWS, XBBBXXBX, ARKKAAKA, QRR, SRR, WEPSRPFPVD, HRRTRKAPKRIRLPHIR, KRTGQYKLGSKTGPGQK, KKTK, KLRSQLVKK, RRRCGQKKK, BX(7)B, RIQNLLKITNLRIKFVK, KKEKDIMKKTI, KGE, RGB, RGBS, TTVVNPKYEGK, ERMSQIKRLLS, WRHRARS, GFOGER, LFDLM, WGREERQ, QSTEKRG, LPNTG, or a combination thereof.

23. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is an internalization domain.

24. The engineered polypeptide of claim 23, wherein the internalization domain is or comprises an amino acid sequence that comprises FXDXF, PPSY, FEDNFVP, YIRV, YADW, YTQV, KKRPKP, SSDDE, RRASS, (YXXL)2, LPLTG, LAFTG, or a combination thereof.

25. The engineered polypeptide of any one of claims 2-13, wherein the targeting domain is a. cell-type specific targeting domain. 188 WO 2021/211467 PCT/US2021/026917

26. The engineered polypeptide of claims 25, wherein the cell-type specific targeting domain is or comprises an amino acid sequence that comprises ASSLNIA, KKEEEKKEEEKKEEE, LIFHKEQ, KFNKPFVFLI, QPEHSST, EYHHYNK, NGR, GEKGEP, KTKKK, KALKKK, KGKKK, CSVTCG, ERE, YKYNLNGRES, YRSL, KGGK7, KKKQYTSIHHG, KDEL, LADQDYTKTA, or a combination thereof.

27. The engineered polypeptide of any one of claims 2-26, wherein the targeting domain is a modified targeting domain in that the targeting domain comprises one or more modified amino acids.

28. The engineered polypeptide of any one of claims 1-27, wherein the nucleic acid release domain is or comprises an amino acid sequence that comprises GRKKRRQRRRPQ, KRH, KSVKKRSVSEIQ, NRRKKRAL, KFERQ, VRGP, NKDS, NRDN, ANNR, or a combination thereof.

29. The engineered polypeptide of any one of claims 1-28, wherein the nucleic acid release domain is a modified nucleic acid release domain in that the nucleic acid release domain comprises one or more modified amino acids.

30. The engineered polypeptide of any one of claims 1-29, further comprising a poly-arginine domain.

31., The engineered polypeptide of claim .30, wherein the poly-arginine domain is a. modified poly-arginine domain in that the poly-arginine domain comprises one or more modified amino acids.

32. The engineered polypeptide of any one of claims 1-31, further comprising a nuclear internalization signal or a nuclear import machinery binding domain. 189 WO 2021/211467 PCT/US2021/026917

33. The engineered polypeptide of claim 32, wherein the nuclear internalization signal or nuclear import machinery binding domain is or comprises an amino acid sequence that comprises KKKYKLK, KKRKLE, TRSK, HRKRKR, NKRKRK, AEKSKKK, RKSK, KRVK, KRK, LQQTPLHLAVL RRPR, PRPR, RPPP, RKKRKGK, PAAKRVKLD, KLKIKRPVK, PKKKRKV, QRKRQK, DSPE, FQVT, QSTEKRG, RQGLID, Cyclic RKKH, or a combination thereof.

34. The engineered polypeptide of claim 32 or claim 33, wherein the nuclear internalization signal or a nuclear import machinery binding domain is a. modified nuclear internalization signal or a nuclear import machinery binding domain in that the nuclear internalization signal or a nuclear import machinery binding domain comprises one or more modified amino acids.

35. The engineered polypeptide of any one of claims 1-34, further comprising a stability domain.

36. The engineered polypeptide of claim 35, wherein the stability7 domain is or comprises an amino acid sequence that comprises YTRF, GDAY, LLEE, RKKRRQRRR, YKSL, YENF, FQDL, YIGSR, IKVAV, or a combination thereof.

37. The engineered polypeptide of claim 35 or 36, wherein the stability domain is a. modified stability domain in that the stability domain comprises one or more modified amino acids.

38. The engineered polypeptide of any one of claims 1-37, further comprising an oligomerization domain.

39. The engineered polypeptide of claim 38, wherein the oligomerization domain is selected from the oligomerization domains of Table 11, optionally wherein the oligomerization domain is positioned at the C-terminus of the engineered poly peptide. 190 WO 2021/211467 PCT/US2021/026917

40. The engineered polypeptide of claim 38 or claim 39, wherein the oligomerization domain is a modified oligomerization domain in that the oligomerization domain comprises one or more modified amino acids.

41. The engineered polypeptide of any one of claims 1-40, wherein the poly peptide comprises a linker.

42. The engineered polypeptide of claim 41, wherein the linker is a linker according to any one of SEQIDNOs: 154-250.

43. The engineered polypeptide of claim 41 or 42, wherein the linker is a modified linker in that the linker comprises one or more modified amino acids.

44. The engineered polypeptide of any one of claims 1-43, wherein one or more amino acids of the engineered polypeptide is a phosphorylated amino acid.

45. The engineered polypeptide of claim 44, wherein the phosphorylated amino acid is a serine, threonine, or tyrosine amino acid.

46. The engineered polypeptide of claim 44 or claim 45, wherein the phosphorylated amino acid, is present in a. linker domain or targeting domain.

47. The engineered polypepti de of any one of claims 1-43, wherein one or more amino acids of the engineered polypeptide is a sulfated amino acid.

48. The engineered polypeptide of claim 47, wherein the sulfated amino acid is a serine, threonine, or tyrosine amino acid.

49. The engineered polypeptide of claim 47 or claim 48, wherein the sulfated amino acid is present in a. linker domain or targeting domain. 191 WO 2021/211467 PCT/US2021/026917

50. The engineered polypeptide of any one of claims 1-43, wherein one or more amino acids of the engineered polypeptide is an acetylated amino acid.

51. The engineered polypeptide of claim 50, wherein the acetylated amino acid is a lysine amino acid.

52. The engineered polypeptide of claim 50 or claim 51, wherein the acetylated amino acid is present in a linker domain or targeting domain.

53. The engineered polypeptide of any one of claims 1-43, wherein one or more amino acids of the engineered polypeptide is a. mannosylated amino acid.

54. The engineered polypeptide of claim 53, wherein the mannosylated amino acid is a. serine amino acid.

55. The engineered polypeptide of claim 53 or 54, wherein the mannosylated amino acid is present in a linker domain or targeting domain.

56. A polynucleotide that encodes the amino acid sequence of an engineered polypeptide of any one of claims 1-55.

57. The polynucleotide of claim 56, wherein the polynucleotide is DMA or RNA.

58. A. vector comprising the polynucleotide of claim 56 or 57.

59. A cell comprising an engineered polypeptide of any one of claims 1-55, a polynucleotide of claim 56 or 57, or a vector of claim 58. 192 WO 2021/211467 PCT/US2021/026917

60. A method of making an engineered polypeptide of any one of claims 1-55, the method comprising expressing a polynucleotide of any one of claims 56-58 in a cell.

61. The method, of claim 60, further comprising isolating the engineered polypeptide from the cell.

62. A. composition comprising: (i) at least one polynucleotide, and (ii) at least one engineered polypeptide of any one of claims 1-55.

63. The composition of claim 62, wherein the at least one polynucleotide is or comprises DNA or RNA.

64. The composition of claim 62 or 63, wherein the at least one polynucleotide comprises a. nucleotide sequence encoding a polypeptide.

65. The composition of any one of claims 62-64, wherein the at least one polynucleotide is or comprises mRNA.

66. The composition of claim 62 or 63, wherein the at least one polynucleotide comprises an inhibitory RNA.

67. The composition of claim 66, wherein the inhibitory RNA is a. gRNA, siRNA, miRNA., or shRNA.

68. The composition of any one of claims 62-67, comprising at least tw7o engineered polypeptides of any one of claims 1-55, wherein a first engineered polypeptide of any one of claims 1-55 is able to oligomerize with a second engineered polypeptide of any one of claims 1- 55. 193 WO 2021/211467 PCT/US2021/026917

69. The composition of any one of claims 62-68, wherein the ratio of polynucleotides to engineered polypeptides of any one of claims 1-55 is between 1:1 and 1:2,000.

70. The composition of claim 69, wherein the ratio of polynucleotides to engineered, polypeptides of any one of claims 1-55 is between 1:1 and 1:1,000, between 1:1 and 1:500, between 1:1 and 1:200, between 1:1 and 1: 100, between 1:1 and 1:50, between 1:3 and 1:1,000, between 1:3 and 1:500, between 1:3 and 1:200, between 1:3 and 1: 100, or between 1:3 and 1:50.

71. The composition of any one of claims 62-70, wherein the ratio of polynucleotides to engineered polypeptides of any one of claims 1-55 is between 1:200 and 1:2,000, between 1:200 and 1:1000, or between 1:200 and 1:500.

72. The composition of any one of claims 62-71, comprising a pharmaceutical carrier.

73. A method comprising administering a composition of any one of claims 62-72 to a system, wherein the system is a cell, tissue, or subject.

74. The method, of claim 73, wherein, after administration, the modification increases the stability, half-life, and/or bioavailability of the composition in the system.

75. The method of claim 73 or 74, 1wherein, after administration, the modification increases the affinity or avidity of the composition with a binding partner in the system, optionally wherein the binding partner is a receptor, cell, or cell membrane.

76. The method of any one of claims 73-75, wherein, after administration, the modification decreases precipitation and/or aggregation of the composition in the system.

77. The method of any one of claims 73-76, wherein, after administration, the modification increases the rate at which the composition enters one or more cells in the system. 194 WO 2021/211467 PCT/US2021/026917

78. The method of any one of claims 73-77, wherein, after administration, the modification increases delivery of the composition to one or more cells in the system.

79. The method, of any one of claims 73-78, wherein, after administration, the modification increases delivery of the nucleic acid of the composition to one or more cells in the system.

80. The method of any one of claims 73-79, wherein the system is a mammalian subject and, after administration, the modification decreases accumulation of the composition in liver.

81. The method of any one of claims 73-80, wherein the system is a mammalian subject and, after administration, the modification increases the amount of composition that crosses the blood-brain barrier.

82. A method comprising administering a. composition of any one of claims 62-72 to a. cell, tissue, or subject, wherein one or more amino acids of the engineered polypeptide is a phosphorylated amino acid.

83. The method, of claim 82, wherein the phosphorylated amino acid is a serine, threonine, or tyrosine amino acid.

84. The method of claim 82 or claim 83, wherein the phosphorylated amino acid is present in a linker domain or targeting domain.

85. The method of any one of claims 82-84, wherein the composition is delivered to cells of the central nervous system (CNS).

86. The method, of any one of claims 82-84, wherein the composition is delivered to CNS neurons. 195 WO 2021/211467 PCT/US2021/026917

87. The method of any one of claims 82-84, wherein the composition is delivered to CNS astrocytes, microglia, oligodendrocytes, or glia.

88. The method, of any one of claims 82-84, wherein the composition is delivered to spinal cord cells, optionally wherein the spina cord cells are spinal cord neurons or spinal cord glial cells.

89. The method of any one of claims 82-88, wherein the polynucleotide encodes an expression product that is expressed in cells to which the composition is delivered.

90. The method of any one of claims 82-89, wherein the subject is a mammalian subject and the administration is intrathecal, intracranial, or intra-cistema magna.

91. A method comprising administering a composition of any one of claims 62-72 to a cell, tissue, or subject, wherein one or more amino acids of the engineered polypeptide is a sulfated amino acid.

92. The method of claim 91, wherein the sulfated amino acid is a serine, threonine, or tyrosine amino acid.

93. The method of claim 91 or claim 92, wherein the sulfated amino acid is present in a linker domain or targeting domain.

94. The method of any one of claims 91-93, wherein the composition is delivered to cells of the central nervous system (CNS).

95. The method of any one of claims 91-93, wherein the composition is delivered to CNS neurons.

96. The method of any one of claims 91-93, wherein the composition is delivered to CNS astrocytes, microglia, oligodendrocytes, or glia 196 WO 2021/211467 PCT/US2021/026917

97. The method of any one of claims 91-93, wherein the composition is delivered to spinal cord cells, optionally wherein the spina cord cells are spinal cord neurons or spinal cord glial cells

98. The method of any one of claims 91-97, wherein the polynucleotide encodes an expression product that is expressed in cells to w-hich the composition is delivered.

99. The method of any one of claims 91-98, wherein the subject is a mammalian subject and the administration is intrathecal, intracranial, or intra-cistema magna.

100. A method comprising administering a composition of any one of claims 62-72 to a cell, tissue, or subject, wherein one or more amino acids of the engineered polypeptide is an acetylated amino acid.

101. The method of claim 100, wherein the acetylated amino acid is a lysine amino acid.

102. The method of claim 100 or claim 101, W'herein the acetylated amino acid is present in a linker domain or targeting domain.

103. The method of any one of claims 100-102, wherein the composition is delivered, to CNS neurons.

104. The method of any one of claims 100-102, wherein the composition is delivered to retinal cells.

105. The method of any one of claims 100-102, wherein the composition is delivered to retinal neurons, optionally wherein the retinal neurons include one or more of photoreceptors, bipolar cells, retinal ganglion cells, horizontal cells, and amacrine cells 197 WO 2021/211467 PCT/US2021/026917

106. The method of any one of claims 100-102, wherein the composition is delivered to photoreceptor cells, optionally wherein the photoreceptor cells include one or both of rods and cones.

107. The method of any one of claims 100-106, wherein the polynucleotide encodes an expression product that is expressed in cells to which the composition is delivered.

108. The method of any one of claims 100-107, wherein the subject is a mammalian subject and the administration is intravitreal, suprachoroidal, or subretinal.

109. A method comprising administering a composition of any one of claims 62-72 to a cell, tissue, or subject, wherein one or more amino acids of the engineered polypeptide is a. mannosylated amino acid.

110. The method of claim 109, wherein the mannosylated amino acid is a serine amino acid.

111. The method of claim 109 or claim 110, wdierein the mannosylated amino acid is present in a linker domain or targeting domain.

112. The method of any one of claims 109-111, wherein the composition is delivered, to CNS neurons.

113. The method of any one of claims 109-111, wherein the composition is delivered to retinal cells.

114. The method of any one of claims 109-111, wherein the composition is delivered to retinal neurons, optionally wherein the retinal neurons include one or more of photoreceptors, bipolar cells, retinal ganglion cells, horizontal cells, and amacrine cells 198 WO 2021/211467 PCT/US2021/026917

115. The method of any one of claims 109-111, wherein the composition is delivered to photoreceptor cells include one or both of rods and cones.

116. The method of any one of claims 109-115, wherein the polynucleotide encodes an expression product that is expressed in cells to which the composition is delivered.

117. The method of any one of claims 109-116, wherein the subject is a mammalian subject and the administration is intravitreal, suprachoroidal, or subretinal.

118. A method of condensing a polynucleotide, comprising contacting the polynucleotide with a polypeptide of any one of claims 1-55.

119. A method of neutralizing the charge of a polynucleotide, comprising contacting the polynucleotide with a. polypeptide of any one of claims 1-55.

120. A composition comprising an engineered polypeptide and at least one nucleic acid, the engineered polypeptide comprising a nucleic acid binding domain and a nucleic acid release domain, wherein one or more amino acids of the engineered polypeptide is a. modified amino acid, optionally wherein the modification comprises at least one of: (i) phosphorylati on, (ii) sulfation; (iii) glycosylation; (iv) prenylation; (v) methylation; (vi) sialylation; (vii) lipidation and/or lipoylation; (viii) acetylation; (ix) hydroxylation; (x) palmitoylation; 199 WO 2021/211467 PCT/US2021/026917 (xi) mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylation; and/or (xv) any combination thereof, optionally wherein the composition is for use in delivering a nucleic acid to a subject or system.

121. The composition of claim 120, wherein the engineered polypeptide comprises a targeting domain.

122. A composition comprising an engineered polypeptide and at least one nucleic acid, the engineered polypeptide comprising a nucleic acid binding domain and a targeting domain, wherein one or more amino acids of the engineered polypeptide is a. modified amino acid, optionally wherein the modification comprises at least one of: (i) phosphorylation, (ii) sulfation; (ill) glycosylation; (iv) prenylation; (v) methylation; (vi) sialyl all on; (vii) lipidation and/or lipoylation; (viii) acetylation; (ix) hydroxylation; (x) palmitoylation, (xi) mannosylation; (xii) myristoylation; (xiii) fucosylation; (xiv) pegylation; and/or (xv) any combination thereof; 200 WO 2021/211467 PCT/US2021/026917 optionally wherein the composition is for use in delivering a nucleic acid to a subject or system.

123. The engineered polypeptide of any one of claims 1-55, the polynucleotide of any one of claims 56-57, the vector of claim 58, the cell of claim 59, the method of any one of claims 60-61, the composition of any one of claims 62-72, 120, 121, or 122, or the method of any one of claims 73-119, wherein the engineered polypeptide has at least 80% sequence identity with an amino acid sequence selected from SEQ ID NOs: 336-388. 201